Restart of DNA replication in Gram-positive bacteria: functional characterisation of the Bacillussubtilis PriA initiator
Nucleic Acids Research
Restart of DNA replication in Gram-positive bacteria: functional characterisation of the Bacillus subtilis PriA initiator
Patrice Polard 1
Stéphanie Marsin 1
Stephen McGovern 1
Marion Velten 1
Dale B. Wigley 0 1
S. Dusko Ehrlich 1
Claude Bruand 1
0 Molecular Enzymology Laboratory, Clare Hall Laboratories, Imperial Cancer Research Fund , Blanche Lane, South Mimms, Potters Bar, Hertfordshire EN6 3LD , UK
1 Laboratoire de Génétique Microbienne, INRA, Domaine de Vilvert , 78352 Jouy en Josas cedex , France
The PriA protein was identified in Escherichia coli as a factor involved in the replication of extrachromosomal elements such as bacteriophage φX174 and plasmid pBR322. Recent data show that PriA plays an important role in chromosomal replication, by promoting reassembly of the replication machinery during reinitiation of inactivated forks. A gene encoding a product 32% identical to the E.coli PriA protein has been identified in Bacillus subtilis. To characterise this protein, designated PriABs, we constructed priABs mutants. These mutants are poorly viable, filamentous and sensitive to rich medium and UV irradiation. Replication of pAMβ1-type plasmids, which is initiated through the formation of a D-loop structure, and the activity of the primosome assembly site ssiA of plasmid pAMβ1 are strongly affected in the mutants. The purified PriABs protein binds preferentially to the active strand of ssiA, even in the presence of B.subtilis SSB protein (SSBBs). PriABs also binds stably and specifically to an artificial D-loop structure in vitro. These data show that PriABs recognises two specific substrates, ssiA and D-loops, and suggest that it triggers primosome assembly on them. PriABs also displays a singlestranded DNA-dependent ATPase activity, which is reduced in the presence of SSBBs, unless the ssiA sequence is present on the ssDNA substrate. Finally, PriABs is shown to be an active helicase. Altogether, these results demonstrate a clear functional identity between PriAEc and PriABs. However, PriABs does not complement an E.coli priA null mutant strain. This host specificity may be due to the divergence between the proteins composing the E.coli and B.subtilis PriA-dependent primosomes.
The Escherichia coli PriA protein was characterised as being
required for replication of bacteriophage φX174 and plasmid
). In these extra-chromosomal elements PriA
promotes the initiation of replication through its specific
binding to DNA, followed by the ordered assembly of several
other proteins, PriB, PriC, DnaT, DnaC, DnaB replicative
helicase and DnaG primase. This particular nucleoprotein complex
has been referred to as the φX174-type primosome (4,5 and
references therein; for recent reviews see 3,6). This primosome
can be sequentially assembled on two distinct DNA sites,
specifically bound by PriA. One, designated pas (primosome
assembly site), was characterised in φX174 and ColE1. PriA
binds to the pas in the single-stranded DNA (ssDNA) form,
when it folds into a particular structure not bound by the SSB
). The second type of PriA binding site is a D-loop
). This three-stranded molecule is an early
intermediate of the replication of ColE1-type plasmids (10). The
cellular function of PriA has emerged more recently, following
the identification of its gene. PriA is not essential in E.coli,
suggesting that initiation of DNA replication promoted by this
protein is accessory (
). However, disruption of the priA
gene decreases cell viability, causes sensitivity to rich medium,
filamentation, UV sensitivity, deficiency in recombination and
constitutive induction of the SOS response. These phenotypes
led to the hypothesis that the cellular role of the φX174-type
primosome is to restart stalled DNA replication, as well as to
repair some types of DNA damage by linking DNA
recombination to replication (
). The structural nature of the
DNA specifically recognised by PriA supports this proposal:
the three-stranded DNA molecules generated by
recombinational repair of the DNA triggers the ordered cascade of
primosomal proteins, inaugurated by PriA, to recruit the DNA
replication machinery (
). To better indicate its cellular
function, the φX174-type primosome has been renamed the
replication restart primosome (17).
In addition to specific binding of DNA, PriA is also a 3′→5′
helicase, translocating along ssDNA in the direction opposite
to the replication fork helicase. This DNA melting activity was
shown to be dispensable for the central role of PriA in E.coli
). Nevertheless, it has recently been proposed that PriA
helicase activity would generate the ssDNA needed for the
loading of DnaB when the forked substrate specifically
targeted by PriA is double stranded (
Current knowledge about primosomal proteins in Bacillus
subtilis is less detailed. Counterparts of the E.coli replicative
helicase and the primase are known in B.subtilis (
there are no obvious homologues of the PriB, PriC, DnaT and
DnaC primosomal proteins (27). Characterisation of the sole
primosome assembly site isolated so far in Gram-positive
bacteria, the ssiA sequence carried by the plasmid pAMβ1, has
pointed to the existence of a φX174-type primosome in
). Three B.subtilis essential proteins, DnaB,
DnaD and DnaI, which are not encoded in the E.coli genome,
are required for chromosomal replication (
) and for ssiA
). A potential PriA analogue was tentatively identified
more recently in B.subtilis on the basis of sequence homology
). Masai et al. purified this protein from an insoluble
fraction and, following renaturation, showed it to be a
DNAdependent ATPase displaying helicase activity and able to bind
to an artificial D-loop structure (
). Nevertheless, this initial
characterisation did not show that the protein was involved in
replication restart in B.subtilis. In this report we address
precisely this question in order to establish the existence of a
PriA-dependent primosome in B.subtilis. We present in vivo
evidence demonstrating functional analogy between this
B.subtilis protein and E.coli PriA. We also report purification
of this protein, which we designate PriABs, in a soluble form,
with which we confirm and extend a previous in vitro study
). More particularly, we report that PriABs displays a much
stronger affinity for ssDNA than its E.coli counterpart.
Altogether, this study confirms the existence of a PriA-dependent
primosome in B.subtilis, built from a conserved initiator.
Finally, we show that PriABs does not substitute for PriAEc
in vivo, suggesting a host specificity for this protein which may
be due to the divergence between the primosomal partners
acting after PriA in the two bacteria.
MATERIALS AND METHODS
Bacterial strains and growth media
The strains used in this study are listed in Table 1. Bacillus
subtilis strains are all derivatives of strain 168. They were
cultivated either in LB medium or in minimal medium (Spizizen’s
minimal salts) (
) supplemented with 0.1% D-glucose, 0.01%
L-tryptophan, 0.1% casamino acids, 18 mg l–1 ammonium
iron(III) citrate (∼17% iron; Merck), as indicated in the text,
and, when required, with 0.6 µ g ml–1 erythromycin (Em), 4 µ g ml–1
chloramphenicol (Cm) and 0.5 or 1 mM IPTG. Competent
cells were prepared as described in Bron (
). The restriction
map of the priABs chromosomal region in strains PPBJ65,
PPBJ69, PPBJ117 and PPBJ120 was verified by Southern
analysis. Strain CBB294 is a derivative of PPBJ120 disrupted
for priABs but carrying a mutation suppressing the lack of
PriABs (dnaB75) (
Plasmid constructions and preparations were done in E.coli
strain MiT898. Escherichia coli strains were grown on Luria
broth supplemented with 25 mg ml–1 thymine or minimal
medium M63 (
) supplemented with 0.2% D-glucose.
Spectinomycin (Spc) (60 µ g ml–1), ampicillin (100 µ g ml–1),
kanamycin (Km) (50 µ g ml–1) and IPTG (for concentrations
see Table 3) were added when required.
Plasmids and M13 derivatives
The plasmids used in this study are listed in Table 1. Plasmids
of the pAPJ series were constructed by inserting various PCR
fragments digested with EcoRI and BamHI between the EcoRI
and BamHI sites of pMUTIN2. These PCR fragments were
generated using chromosomal DNA of strain 168 as template
and the following oligonucleotides as primers: pAPJ11, OPP8
and OPP9; pAPJ12, OPP10 and OPP11; pAPJ13, OPP12 and OPP14;
pAPJ14, OPP13 and OPP14. OPP8,
5′-CCGGAATTCGCTCTGTAACCATCAAAACCC-3′ (+689 to +710); OPP9,
5′-CGCGGATCCGAAGCGGCCCTTGAAGCGG-3′ (+1023 to +1002);
(+2079 to +2098); OPP11,
5′-CGCGGATCCTTACATCATCATATAAGG-3′ (+2419 to +2401); OPP12,
5′-CCGGAATTCTCAAAACAAACCGGAGAGCGC-3′ (–25 to –3); OPP13,
5′CCGGAATTCAATTTTGCAGAAGTCATCGTTG-3′ (+4 to
5′-CGCGGATCCGGAAGATCGGCTCCGTGTGC-3′ (+376 to +355). The priABs sequence is italicised and
the EcoRI and BamHI sites are underlined. Arbitrary
coordinates for the priABs sequence retained in the oligonucleotides
are indicated in parentheses, giving the value +1 to the A of the
proposed translational start of the ORF (
). The sequence of
the insert in pAPJ13 has been checked to ascertain the absence
For construction of pSMG3, the PriABs coding sequence was
PCR amplified from chromosomal DNA of strain 168 with
OPP17 and OPP18 as primers and inserted into the NdeI and SapI
sites of pCYB1 (both blunted by Klenow filling in): OPP17,
5′-CATCATCATATAAGGATTCATATC-3′. This generated a triple
fusion protein, PriABs–intein–chitin binding domain (CBD),
expressed under the control of E.coli transcriptional (the Ptac
promoter, inducible by IPTG) and translational signals. The
sequence of priABs carried by pSMG3 has been verified.
For construction of pSMG19, the SSBBs coding sequence
was PCR amplified from chromosomal DNA of strain 168
with OSMG18 and OSMG19 as primers, digested with NdeI and
SapI and inserted in the sames sites of pTYB1: OSMG18,
5′-GAATTCGCTCTTCCGCAGAATGGAAGATCATCATCCGAGATG-3′ (sequences underlined in OSMG18 and
OSMG19 represent a NdeI and a SapI site, respectively). This
generated a triple fusion protein SSBBs–intein–CBD, expressed
under the control of transcriptional and translational signals of
bacteriophage T7. The sequence of ssbBs carried by pSMG19
has been verified.
pAPJ41 is a derivative of the pGB2 vector (
) which allows
inducible expression of PriABs in E.coli and which does not
need PriAEc for replication. This plasmid was constructed in
several steps. First, the chromosome of strain PPBJ69 was
digested with SwaI, ligated and transformed into E.coli to
isolate a plasmid, pAPJ19, which contains the whole priABs
ORF and additional 3′-flanking sequences. Second, the 3′ end
of the priABs–intein–CBD ORF in pSMG3 (SacI–BamHI) was
exchanged for the 3′ end of the priABs ORF of pAPJ19
(SacI–BglII) to generate plasmid pSMG4. Finally, to obtain
pAPJ41 the Eco47III–PstI restriction fragment of pSMG4
carrying the laciq gene and the priABs ORF placed under the
control of the Ptac promoter was cloned in the pGB2 vector
between the SmaI and PstI sites located in the polylinker.
pAPJ43 is almost identical to pAPJ41 except that it carries the
PriAEc coding sequence in place of PriABs. It was constructed in
two steps. First, the NdeI–PvuI fragment of the PriAEc-expressing
plasmid described in Nurse et al. (
) was exchanged with a
similarly cleaved fragment of pSMG3 to give plasmid pAPJ42.
Then the MluI–HindIII fragment of pAPJ42 carrying the whole
Ptac–priAEc artificial gene was exchanged for the corresponding
MluI–HindIII fragment of pAPJ41 to give pAPJ43.
The two M13mp19 derivatives carrying the ssiA sequence in
both orientations, M13-ssiA+ and M13-ssiA–, were constructed in
two steps. The ssiA sequence (145 nt, coordinates 4712–4856 in
) was generated by PCR using pIL253 as template
and OPP1 and OPP3 as primers, and inserted in both orientations
in the SmaI site of the polylinker of plasmid pAPJ2, giving
plasmids pAPJ9 and pAPJ10: OPP1,
5′-CCTATAAAAGATAGAAAATTAAAAAATC-3′. The sequence of ssiA has been
verified by DNA sequencing. The activity of ssiA has been
verified in B.subtilis by showing that the ssDNA of plasmid
pAPJ9 is efficiently converted into double-standed DNA
(dsDNA) in vivo. Finally, the SalI–EcoRI fragments carrying
ssiA from pAPJ9 and pAPJ10 were cloned into M13mp19
similarly cut, to give M13-ssiA+ and M13-ssiA–, respectively.
Plating efficiency and UV survival tests
Bacillus subtilis strains were grown to mid-log phase in
minimal medium containing Em with and without IPTG
(1 mM) as indicated (see legend to Fig. 2 and Table 2). To
measure the plating efficiency, cultures were diluted
appropriately and plated on minimal medium and on LB similarly
supplemented with Em and IPTG, and incubated for 16–40 h at
37°C. To measure UV survival, minimal medium plates were
irradiated immediately after plating with a 2 J m–2 dose of UV
for different time periods and incubated for 16–40 h at 37°C.
The tests with E.coli were performed similarly. Strains were
grown in minimal medium containing Spc, supplemented or
not with IPTG, as indicated (see Table 3), and spread on plates
not supplemented with IPTG.
DNA manipulation and analysis
Standard techniques were used for DNA manipulation and
cloning in E.coli (
). Total DNA from exponentially growing
B.subtilis cells was extracted as described (
). After agarose
gel electrophoresis, plasmid DNA was revealed by Southern
blotting with α-32P-radiolabelled probe generated with a nick
translation kit (Roche) with purified plasmid DNA in the
presence of [α-32P]dATP (ICN). The different plamid species were
revealed and quantified with a Storm apparatus (Molecular
Dynamics) and ImageQuant software.
DNA sequencing of PCR products or plasmid templates was
done with the PRISM sequencing kit (Applied Biosystems)
and resolved on an automated DNA sequencer (Applied
M13 ssDNA was prepared from TG1 cells as described (
pAPJ9 ssDNA was similarly prepared from TG1 cells
containing the helper phage M13K07 (
DNA probes used for the electrophoretic mobility shift
assays were prepared by several means. The 174 nt long ssiA+/–
ssDNA probes were excised by restriction from M13-ssiA+ and
M13-ssiA– ssDNA with the use of the following
oligonucleotides complementary to restriction sites flanking ssiA:
OSMG42, 5′-CGTCGACCTGCAGCATGCA-3′; OSMG43,
5′-GCCGATGAATTCGATTAAT-3′. The underlined sequences represent a PstI
site in OSMG42 and an EcoRI site in OSMG43 and OSMG44. The
OSMG42/OSMG43 pair was used to excise ssiA+ from the
M13-ssiA+ ssDNA template and, similarly, the OSMG42/OSMG44
pair to excise ssiA– from M13-ssiA–. The ssDNA (50 nM) was
heated at 65°C for 10 min in a 1 ml solution containing
100 mM NaCl, 20 mM Tris, pH 7.5, 1 mM DTT and 10 mM
MgCl2 and the complementary pair of oligonucleotides
(500 nM) were allowed to anneal by cooling the mixture
slowly to 37°C. The DNA was then digested to completion
with PstI and EcoRI. The 174 nt fragments released were
purified from the larger bacteriophage DNA fragment and
smaller oligonucleotides by gel filtration on Superose 6
(Pharmacia). They were then treated with shrimp
phosphatase (Pharmacia) before 5′-end-labelling using
[γ-32P]ATP (ICN) and T4 polynucleotide kinase. Finally, both
fragments were purified by electrophoresis on 5% (w/v)
polyacrylamide gels and recovered by passive elution in buffer E
(10 mM Tris, pH 8, 1 mM EDTA, 0.2% SDS, 0.3 M NaCl)
overnight at 30°C. The size and uniformity of the fragments was
verified on a denaturing polyacrylamide gel using a sequence
dsDNA and branched DNA molecules were prepared by
annealing the following purified oligonucleotides, which are
identical to those used by McGlynn et al. (
) for the study of
PriAEc and RecG binding to D-loop and bubble structures:
5′-AAAGATGTCCTAGCAAGGCAC-3′. The D-loop was made by
annealing OSMG27, OSMG28 and OSMG29 mixed at a molar ratio of
1:2:3, respectively. The bubble was made by annealing OSMG27
and OSMG28 and the dsDNA with OSMG27 and OSMG27comp at a
molar ratio of 1:2. Annealing was performed by heating the
oligonucleotides in buffer A (10 mM Tris, pH 8, 1 mM EDTA,
100 mM NaCl) for 5 min at 95°C, then they were left for
10 min at 65°C, followed by slow cooling to room temperature. In
each combination, the OSMG27 oligonucleotide was 5′-end-labelled
with [γ-32P]ATP (ICN) and T4 polynucleotide kinase prior to
annealing. The expected synthetic DNA substrates were
purified by elution in buffer E after separation from free
oligonucleotides by native electrophoresis in a 5% polyacrylamide
gel. The Ost4 oligonucleotide was used as a ssDNA probe,
prepared as for the other DNA substrates: Ost4,
For each probe, the concentrations of DNA substrates used
in the gel mobility assay were estimated by monitoring the
specific activity of the labelled oligonucleotide after end labelling
and the final activity of the purified substrate.
Purification of PriABs, PriAEc and SSBBs
PriABs and SSBBs were expressed and purified using the
IMPACT system (New England Biolabs). PriABs and SSBBs
proteins fused to the intein–CBD tag were overproduced in
strains MiT898 and B834 (DE3), respectively. To limit protein
aggregation, cell growth was carried out at 25°C. Optimal
conditions for PriABs production were IPTG induction for 3 h
at the end of exponential growth, and for SSBBs production
overnight growth without induction. Cells were harvested,
resuspended in HEN500-T buffer (20 mM HEPES pH 7.6,
0.1 mM EDTA, 500 mM NaCl, 0.1% Triton X-100) and
broken by sonication (Bioblock Vibracell 72408 sonicator,
used as recommended by the supplier). The lysate was
centrifugated at 4°C for 1 h at 20 000 g, the supernatant loaded onto
chitin beads and the protein separated from intein by addition
of 30 mM DTT and incubation overnight at 4°C. The protein
was eluted and further purified by conventional
chromatography. In the case of PriABs, the protein in HEN100-D buffer
(20 mM HEPES pH 7.6, 0.1 mM EDTA, 100 mM NaCl, 1 mM
DTT), was loaded onto a Hi-Trap SP-Sepharose column
(Pharmacia) and eluted with a linear NaCl gradient in HED
buffer. The fractions containing PriABs were loaded onto a
Hi-Trap heparin column (Pharmacia) and the protein bound
was eluted with HEN100-D. It was diluted twice with 100%
glycerol and stored at –20°C. The yield of PriABs was ∼2 mg
protein l–1 of culture and its purity was estimated to be 95%. In
the case of SSBBs, the protein eluted from the chitin beads was
further purified by successive chromatography on Hi-Trap
Q-Sepharose and heparin columns (Pharmacia). The eluted
proteins were finally treated by heat (at 85°C for 5 min) to
eliminate by precipitation the contaminants which co-purify
with SSBBs. We have shown that such a heat treatment does not
modify SSBBs binding activity to ssDNA, as shown previously
for SSB of E.coli (
). We have also observed by gel filtration
on a Superose 12 column (Pharmacia) that purified SSBBs is a
tetrameric protein, like its E.coli counterpart. The yield of
SSBBs was ∼0.5 mg protein l–1 and its purity was estimated to
The same purification procedure was used for PriAEc as for
PriABs, except that expression was in strain JC19008 carrying
plasmid pSMG24 (
). The yield of purified soluble protein
was similar in both cases.
Immunodetection of PriABs
Immunisation against PriABs and serum preparation in the
rabbit was entrusted to Eurogentec. Prior to injection, PriABs
protein was further purified to homogeneity by electrophoresis
on a SDS–polyacrylamide gel. The antibodies directed against
PriABs were purified from serum by the method described in
Pringle et al. (
) after coupling PriABs to Affi-Gel10 as
recommended by the supplier (Bio-Rad). The relative levels of
PriABs in the different strains used were determined by
immunoblot analysis with the purified antibodies. Cells were
grown exponentially in LB medium suplemented with IPTG.
Cell lysates were prepared by lysozyme treatment of the
harvested cells followed by brief sonication. The same
amounts of total cellular proteins of each strain were then
fractionated by SDS–PAGE on 8% gels and transferred to a
Hybond PVDF membrane (Amersham) by electroblotting
using a semi-dry transfer system. PriABs immunodetection was
carried out as described in the ECL+ kit (Amersham). Purified
anti-PriABs antibodies were diluted 1/500 for the hybridisation
step. Protein G–horseradish peroxidase (Bio-Rad) was used to
reveal anti-PriABs with a Storm apparatus (Molecular
Dynamics) and quantification was with ImageQuant software.
Gel mobility shift assays
For some experiments reported (see Figs 4 and 5) the reaction
mixture (40 µ l) contained 10 mM HEPES pH 7.5, 3 mM DTT,
200 mM NaCl, 0.2 mg/ml bovine serum albumin (BSA) and
γ-32P-labelled DNA, at the concentrations specified in the
figure legends. The amounts of purified PriABs and SSBBs
(expressed in nM) are indicated in the figures. Reaction
mixtures were incubated at 30°C for 15 min and analysed by
gel electrophoresis through a 5 or 4% (80:1) polyacrylamide
gel as indicated in the figure legends, following addition of
10 µ l of 50% glycerol (supplemented with 0.04% xylene
cyanol and 1 mg ml–1 BSA). In the case of the binding
experiments performed with SSBBs and PriABs, SSBBs was
pre-incubated for 15 min at 30°C with the ssDNA substrates
prior to addition of PriABs, which was then allowed to further
interact for 15 min at 30°C. For other experiments (see Fig. 6),
various amounts of PriABs and PriAEc were incubated with
labelled DNA substrates (0.1 nM) in 20 µ l of R buffer (50 mM
HEPES, 1 mM DTT, 1 mM EDTA, 0.1 mg ml–1 BSA, 50 mM
NaCl, 12.5% glycerol, pH 7.4) at 30°C for 10 min. At the end
of incubation, 5 µ l of loading buffer (50% glycerol, 0.4%
cyanol, 0.1 mg ml–1 BSA) was added and the samples were
loaded on a 5% polyacrylamide gel (30:1) containing 5%
glycerol and 0.25× TBE. Three different electrophoresis
buffers were used: TAM (6 mM Tris, 5 mM Na acetate, 2 mM Mg
acetate) (see Fig. 5), TEG (25 mM Tris, 0.19 M glycine, 1 mM
EDTA) (see Figs 4 and 5) or 0.25× TBE (90 mM Tris–borate,
2 mM EDTA) (see Fig. 6). Electrophoresis was at 4°C at
12 V cm–1 for 2–4 h with circularisation of the buffer. Following
electrophoresis, gels were dried under vacuum, revealed with a
Storm apparatus (Molecular Dynamics), the radioactivity
quantified with ImageQuant software and the apparent Kd
determined according to Riggs et al. (
ATPase activity was assayed by linking ATP hydrolysis to the
oxidation of NADH as described previously (
dependence of the ATPase reaction on ssDNA cofactor was examined
by the above method at 37°C in a buffer containing 50 mM
HEPES pH 7.5, 50 mM NaCl, 5 mM MgCl2, 0.2 mg ml–1 BSA.
The concentrations of DNA (expressed in nM nt), ATP and
proteins PriABs and SSBBs (in nM) used in the assays are
indicated in the figure legends, as well as the time course of the
reaction. Kinetic experiments were performed in a UV/VIS
spectrometer Lambda 20 (Perkin Elmer). Values for the
Michaelis–Menten constants kcat and Km for ATP at saturating
amounts of ssDNA were derived by fitting data directly to the
The same standard forked DNA used for study of the Thermus
aquaticus helicase (
) was used to assay the helicase activity
of PriABs. It was similarly prepared by annealing the following
two purified oligonucleotides, after labelling of the 5′ end of
oligonucleotide OPP210 with T4 polynucleotide kinase (NEB):
OPP210, 5′-T30CGAGCACCGCTGCGGCTGCACC-3′; OPP211,
Helicase assays were performed with the indicated amount
of PriABs added to 1 nM DNA substrate in 20 µ l of reaction
buffer composed of 20 mM Tris pH 7.5, 50 mM NaCl, 3 mM
MgCl2, 4 mM DTT, 20 µ g ml–1 BSA, with or without addition
of ATP (5 mM) as indicated. After 30 min incubation at 30°C
each reaction was stopped with 5 µ l of S solution (3% SDS,
100 mM EDTA, 40% glycerol, 0.1% xylene cyanol) and run
through a 12% polyacrylamide gel in 1× TBE. Following
electrophoresis, gels were dried under vacuum, revealed with a
Storm apparatus (Molecular Dynamics) and the radioactivity
present in the forked substrate and in the ssDNA product
quantified with ImageQuant software to calculate the level of
helicase activity expressed as a percentage of ssDNA
generated in the assay.
Bacillus subtilis encodes a homologue of the E.coli
primosomal PriA protein
The sequence of a B.subtilis ORF encoding a homologue of
PriAEc has been reported (
). This ORF, designated priABs,
is located at 140° on the B.subtilis map. On the basis of
sequence analysis, priABs is the second ORF of an operon
including 12 ORFs. The level of homology of PriABs with the
E.coli protein is highly significant, with 32% identity and 65%
similarity distributed along the two proteins. However, PriABs
contains 70 additional amino acids, clustered in the first third
of the protein. Two distinct functional regions present in PriAEc
are conserved in the B.subtilis protein. One corresponds to the
seven canonical motifs typical of many helicases and the other
to a cysteine-rich region, which may be organised in two
consecutive small zinc finger-like motifs (Fig. 1A).
To show that priABs is transcribed and translated, its putative
product has been overproduced in E.coli and purified (Fig. 1B)
and specific antibodies directed against this protein have been
prepared and purified (see Materials and Methods). Whole
cellular protein extracts from B.subtilis were analysed by
western blotting using the anti-PriABs antibodies (Fig. 1C). A
protein identical in size with the purified PriABs was detected
in the B.subtilis wild-type strain (Fig. 1C, lane 5). The specifity
of the signal was demonstrated by its disappearance in strain
CBB294, in which priABs has been disrupted (see below;
Fig. 1C, lane 8). Therefore, PriABs is expressed in B.subtilis.
The number of PriABs molecules per cell is between 50 and
100, as deduced from the western blot analysis.
Bacillus subtilis priA mutants are poorly viable, sensitive to rich medium and UV irradiation
To study the role of PriABs in cell physiology, we constructed
priABs mutants. For this purpose we disrupted priABs by
transforming B.subtilis 168 cells with non-replicative EmR plasmids
carrying internal fragments of priABs (pAPJ11 and pAPJ14). As
a control, we used a plasmid carrying the 3′ end of priABs, and
thus expected to preserve the integrity of priABs upon insertion
(pAPJ12). As E.coli null priA mutants are viable on minimal
medium but not on rich medium (
), disruption was carried
out on minimal medium. EmR transformants were obtained
with the disrupting plasmids, giving strains PPBJ117 and
PPBJ120, which carried priABs alleles designated priA2Bs and
priA1Bs, respectively (Fig. 1D). However, colonies were much
smaller in size than upon transformation with the control
plasmid. As expected from the size of the colonies, these
strains grow slowly in minimal medium (doubling time
>160 min) and microscopic examination of the bacteria
revealed that they were filamentous. Measurements of the
plating efficiencies of these strains showed that they were
40–100-fold less viable than the control strain on minimal
medium and were sensitive to rich medium (Table 2), as well
as to UV irradiation (Fig. 2). A strain in which priABs is under
the control of the Pspac promoter was also constructed (PPBJ69
carrying the priAind allele) (Fig. 1D). This strain displayed
wild-type phenotypes in the presence of IPTG (see Table 2 and
Fig. 2), although its level of PriABs was 3-fold lower than in the
control strains, as shown by western blot analysis (Fig. 1C,
aSee Figure 1D.
bThe strains were grown to mid-log phase in minimal medium in the presence
(+) or absence (–) of IPTG (1 mM). Appropriate dilutions of the cultures were
plated on either rich or minimal medium containing IPTG. c.f.u., colony
forming units. Values are the average of between two and four independent
determinations, except for PPBJ69 in rich medium + IPTG, which was tested only
compare lanes 5 and 6 with 7). In the absence of IPTG,
however, this strain displayed phenotypes similar to those of
disrupted strains: filamentation, small colonies and poor
viability in minimal medium, as well as sensitivity to rich
medium (Table 2) and sensitivity to UV irradiation (Fig. 2).
PriABs is required for primosome assembly on ssiA and
D-loops in vivo
In E.coli, PriA was initially characterised as being required for
two distinct modes of replication displayed by extrachromosomal
elements. The first relies on a pas sequence required for
replication of the ssDNA circular intermediate generated during
rolling circle replication of bacteriophage φX174 (
second depends on a D-loop structure synthesised at an early
step of the θ replication mode of ColEI-type plasmids (
Interestingly, these two schemes of replication have been
characterised in B.subtilis and have been shown to rely on
identified primosomal proteins of this bacterium (
). Therefore, we
tested PriABs dependence for these two modes of
extrachromosomal replication. The pas-mediated conversion of ssDNA to
dsDNA was measured in a plasmid rolling circle assay with the
use of the pas sequence ssiA from plasmid pAMβ1 (
Dloop-mediated replication was measured using an appropriate
pAMβ1 derivative (
). The experiments were carried
out in strains harbouring the conditional priAind allele,
allowing modulation of priABs expression with the use of IPTG
To test the activity of ssiA, we used derivatives of the rolling
circle plasmid pC194, which produce a ssDNA intermediate
which is not efficiently converted to the dsDNA form in
). This circular ssDNA molecule is detected by
Southern blotting following electrophoresis of total DNA
prepared from B.subtilis cells harbouring such plasmids
(Fig. 3A). In the PriA+ strain PPBJ65, conversion of ssDNA to
the dsDNA form is promoted by ssiA in the active orientation
(ssiA+) but not in the inactive orientation (ssiA–) (Fig. 3A, lanes
3 and 1). In contrast, in the priAind strain, conversion is
inefficient, irrespective of the ssiA orientation. Conversion was
inefficient both in the absence (Fig. 3A) and presence of IPTG (not
shown), indicating that the diminished level of PriABs in the
induced priAind strain is not sufficient to support ssiA activity
on a multicopy extrachromosomal element, while it appears
sufficient for chromosomal replication.
pAMβ1-type plasmids replicate by a θ mechanism that
involves an early D-loop intermediate, in which the ssiA+
sequence is present on the ssDNA portion of the molecule
). In the priAind mutant grown without IPTG the copy
number of plasmid pVA798∆ RCR (a pAMβ1-type plasmid;
48) was ∼10-fold lower than in PriABs+ cells and the plasmid
accumulated ssDNA (Fig. 3B). The ssDNA corresponded to
the plasmid lagging strand template, as demonstrated by the
use of strand-specific probes (not shown). A similar replication
defect was observed when the cells were grown in the presence
of IPTG (not shown), presumably reflecting the low PriABs
level in the cells. The defect of pVA798∆ RCR replication in
the absence of PriABs led to loss of the plasmid from priAind
cells upon prolonged growth without IPTG and to its inability
to become established in the priA1Bs strain (not shown). We
conclude that PriABs is required for replication of pAMβ1-type
plasmids and acts presumably by promoting primosome
assembly on the D-loop intermediate. The ssiA sequence
unmasked on the D-loop was previously shown not to be
essential for pAMβ1 replication, suggesting a
ssiA-independent mechanism(s) of primosome assembly (
priAind mutants pAMβ1 derivatives lacking ssiA exhibited a
reduced copy number and accumulated ssDNA (not shown).
We conclude that a ssiA-independent mechanism(s) of
primosome loading during pAMβ1 replication is dependent on
PriABs and assume that PriABs, like PriAEc, triggers primosome
assembly on D-loops.
PriABs binds preferentially to the primosome assembly site
Analysis of ssDNA conversion to dsDNA indicated that PriABs
acts in vivo at ssiA. In order to test whether PriABs recognises
the active strand of ssiA, we carried out gel shift analyses with
the purified protein. For these experiments two 174 nt long
ssDNA substrates, carrying either the active or inactive strand
of ssiA (designated ssiA+ and ssiA–, respectively), were
prepared and radiolabelled (see Materials and Methods).
Somewhat surprisingly, the two substrates behaved differently
in non-denaturing gels: the ssiA+ strand migrated faster than
the ssiA– strand (Fig. 4A, lanes 1 and 6) and was accompanied
by a minor, slowly migrating product (Fig. 4A, lane 1). In
contrast, the two strands appeared identical in size in a gel
under denaturing conditions (data not shown). This suggests
that the two strands might fold into different secondary or
tertiary structures. Binding experiments conducted with
B.subtilis SSB protein (SSBBs; see Materials and Methods for
purification procedure) led to the same conclusion. Both
strands were efficiently recognised by SSBBs, but the binding
patterns were clearly different (Fig. 4A). The faster migrating
form of ssiA+ gave primarily two shifted bands while ssiA–
gave an additional band at saturating amounts of SSBBs. The
slower ssiA+ form gave multiple shifted bands. This indicates
that a part of the ssiA+ sequence may be poorly accessible to a
PriABs binding to the ssiA substrates indicated a 7-fold
preference for ssiA+ (Kd = 0.45 nM) over ssiA– (Kd = 3.1 nM)
(Fig. 4B). PriABs generated one complex with the ssiA+
substrate at low concentration and up to three complexes at
higher concentrations. The slow migrating form of ssiA+ was
also shifted by PriABs to poorly resolved multiple bands. These
experiments revealed that PriABs can bind stably to the inactive
ssiA strand. This interaction does not depend on the presence
of the ssiA sequence since it occurs with any ssDNA molecule
longer than 41 nt, but not with dsDNA, to which PriABs binds
poorly (data not shown).
Finally, we carried out a binding experiment between PriABs
and ssiA strands preincubated with an excess of SSBBs. Under
these conditions only the ssiA+ substrate was shifted by PriABs
(Fig. 4C). Both fast and slow migrating forms of ssiA+ were
shifted, indicating that they have related structures (Fig. 4C).
PriABs binds specifically to a D-loop structure
Analysis of pAMβ1-related plasmids indicated that PriABs acts
in vivo at D-loops (see above). We therefore investigated
whether PriABs binds to the D-loop and bubble structures used
previously to reveal PriAEc binding (
). As shown in Figure 5,
PriABs bound preferentially to the D-loop (Kd = 1.5 nM) in
comparison to a bubble (Kd = 150 nM). Three complexes (DI,
DII and DIII) (Fig. 5A) appeared consecutively with increasing
amounts of PriABs added to the D-loop, whereas only two were
observed with the bubble (BI and BII) (Fig. 5B). The
appearance of DI at low protein concentrations, followed by that of
DII, DIII, BI and BII at higher concentrations, clearly
demonstrates the preferential binding of PriABs to the D-loop
structure. A small amount of unbound contaminant bubble structure
in the D-loop preparation (Fig. 5A, band B) provided an
internal control which confirmed the preference for the D-loop.
Furthermore, complexes of PriABs with the bubble substrate
(BI and BII) were much less stable than those generated with
the D-loop, since they almost completely disappeared when
electrophoresed under destabilising conditions (i.e. in the
presence of magnesium; Fig. 5D), whereas complexes with the
D-loop remained stable (Fig. 5C). These results confirmed and
detailed what was previously reported with PriABs purified
differently from an insoluble form (
PriABs binds strongly to ssDNA
The above gel shift experiments indicated that PriABs displays
a high affinity for ssDNA, which would clearly distinguish
PriABs from its functional homologue PriAEc. To compare the
two proteins with respect to their ssDNA binding activity, we
performed gel shift experiments with the ssDNA substrate
Ost4, a 90 nt long oligonucleotide. The PriAEc protein was
purified by a procedure similar to that used for PriABs (see
Materials and Methods). As shown in Figure 6, PriABs binds
with a better affinity than PriAEc to the ssDNA probe (compare
Fig. 6A and B, left; Kd = 4 and 20 nM, respectively). PriABs
generated two discrete retarded bands, while a smear from the
retarded band to the free DNA was observed with PriAEc. Such
a gel shift pattern is strongly indicative that PriAEc binding to
ssDNA is unstable. Accordingly, increasing the ionic strength
of the binding buffer nearly eliminated the band shift induced
by PriAEc, while PriABs still bound to the substrate efficiently
under those conditions (compare Fig. 6A and B, right).
PriABs is a ssDNA-dependent ATPase displaying helicase
PriAEc is a ssDNA-dependent ATPase fueling its helicase
). We have observed that PriABs induces ATP
hydrolysis in the presence of naked ssDNA, but not dsDNA
(Fig. 7A), indicating that it has a similar activity. Stable
binding of PriABs to ssDNA is not required for this activity,
because a 21 nt long oligonucleotide efficiently triggered
PriABs ATPase activity (Fig. 7A) but did not form a stable
complex with PriABs as judged by gel shift experiments (data
not shown). The presence of ssiA+ or ssiA– in naked M13
circular ssDNA did not affect the ATPase activity of PriABs
(Fig. 7B). However, addition of SSBBs protein prior to PriABs,
which reduced ATPase activity with all substrates, had less
effect with the ssiA+ than with the ssiA– substrate (Fig. 7C).
We conclude that PriABs is a ssDNA-dependent ATPase and
suggest that SSBBs limits the accessibility of PriABs to
ssDNA, unless a specialised DNA sequence, such as ssiA, is
present in the ssDNA. The ATPase activity of PriABs is linked
to the translocase/helicase activity already reported for PriABs
). As illustrated in Figure 7D, we have also confirmed that
our PriABs preparation displayed helicase activity: provided
that ATP was included in the reaction, PriABs efficiently
unwound a Y-shaped DNA molecule used in the helicase assay
PriABs does not complement an E.coli priA null mutant
The results presented above show that PriABs is functionally
equivalent to PriAEc in vivo and in vitro, as was previously
proposed by Masai et al. (
). A question raised by this
identity is whether one PriA can substitute for the other in vivo. We
tested this hypothesis with PriABs in E.coli. For this purpose we
cloned priABs in pGB2, a plasmid which does not depend on
PriAEc for replication, and placed it under the control of E.coli
translational and IPTG-inducible transcriptional signals to give
plasmid pAPJ41 (see Materials and Methods). Another
plasmid, pAPJ43, carrying the priAEc coding sequence under
the same expression signals, was constructed as a control. The
priAEc null mutant strain JC18983 and the wild-type isogenic
strain DM4000 (
) were transformed by the two plasmids and
tested for several phenotypes associated with the lack of
PriAEc: viability, growth on rich medium, UV sensitivity and
replication of the ColE1-type plasmid pBR322. As expected,
the priAEc-carrying plasmid corrected the phenotypes of the
priA mutant even without IPTG induction (Table 3) (only
viability and sensitivity to rich medium were tested for this
plasmid). In contrast, the priABs-carrying plasmid did not
correct any of the mutant phenotypes, either non-induced or
induced with low IPTG concentrations (Table 3). At higher
IPTG concentrations induction of PriABs was toxic (Table 3).
The toxicity associated with PriABs expression was not
observed in the wild-type strain (Table 3). These results show
that PriABs cannot substitute for PriAEc in vivo.
We report a detailed analysis of a B.subtilis protein proposed to
be the counterpart of the E.coli PriA primosomal protein on the
basis of sequence similarities (
), confirming and
extending a previous biochemical analysis of this protein (
Several lines of in vivo and in vitro evidence demonstrate that
this protein is indeed PriA.
Typical phenotypes associated with the lack of PriA in E.coli
have been observed with B.subtilis priA mutant cells. These
include poor viability, slow growth, filamentation and
sensitivity to rich medium and UV. In E.coli these defects of priA
mutants are thought to be due to a deficiency in the repair of
arrested replication forks (for reviews see 6,16,17). We
therefore propose that PriABs plays a similar role in replication fork
reactivation in B.subtilis.
c.f.u. × 108 per OD600a Growth on rich UV resistancec pBR322
c.f.u., colony forming units; ND, not determined.
aStrains were grown exponentially at 37°C with the indicated dose of IPTG in minimal medium supplemented with spectinomycin. At OD600 = 0.4–0.6 cells were
serially diluted, plated on minimal media without IPTG and with spectinomycin and c.f.u. were determined after 48 h incubation at 37°C.
bCells grown in minimal medium without IPTG and with spectinomycin were streaked onto LB plates supplemented with spectinomycin and the indicated dose of
IPTG, and incubated at 37°C for 48 h. + and – indicate the presence and absence of colonies, respectively.
cAs in footnote a except that after plating, cells were irradiated at 20 J m–2. Results are expressed as the fraction of surviving cells.
dAbility of pBR322 to replicate was measured by its ability to transform competent cells to tetracyclin resistance. +, ∼106 transformants µ g–1; –, <100 transformants µ g–1.
PriAEc is required for replication of several E.coli
extrachromosomal elements (
). We report defects in two modes of
extrachromosomal replication in B.subtilis priA mutants. One
is the ssiA-dependent conversion of ssDNA to dsDNA. ssiA
has been shown to act as a primosome assembly site in
), similarly to the pas sequence of bacteriophage
φX174 in E.coli (
). Moreover, we show that PriABs binds
stably and specifically to the active strand of ssiA in vitro, as
does the E.coli protein to the pas sequence of φX174 (
PriABs still binds to ssiA in the presence of SSBBs protein.
These results show that PriABs binds ssDNA carrying ssiA and
suggest that it triggers primosome assembly at this site.
Interestingly, ssiA appears to adopt a particular structure while its
complementary strand does not. It has been shown that pas
sites in E.coli are structurally distinct from their complementary
strands and are resistant to melting by SSB and it is proposed
that this feature determines their recognition by PriAEc (
Similarly, we propose that the structure adopted by ssiA is
refractory to melting by SSBBs and that this contributes to its
specific recognition by PriABs in vivo.
Another mode of extrachromosomal replication dependent
on PriABs is that of the pAMβ1-type plasmids, which is similar
to that described for the E.coli ColE1-type replicons (
involves the formation of a D-loop structure, to which PriAEc
binds specifically and promotes primosome assembly in vitro
). PriABs protein efficiently binds an artificial D-loop
structure in vitro (this study; 31). We observed that PriABs
interaction with this three-stranded DNA molecule is more
specific and much more stable than with a bubble structure.
These combined in vivo and in vitro analyses suggest that
PriABs triggers primosome assembly on such branched
molecules, as reported for PriAEc. Such structures are thought
to be targeted by PriA during DNA recombinational repair
Another characteristic shared by the B.subtilis and E.coli
PriA proteins is their low quantity in the cell, estimated to be
50–100 molecules per cell (
). We present evidence that this
level can be reduced 3-fold in B.subtilis without the
appearance of detectable cellular defects. However, the diminished
quantity of PriABs is not high enough to sustain a normal level
of PriABs-dependent extrachromosomal replication.
During the course of this study the purification of PriABs, a
description of its binding to D-loop structures and its
ssDNAdependent ATPase and helicase activities have been reported
). Our in vitro observations with a purified soluble form of
PriABs confirm and extend this preliminary report. We have
observed a more stable binding of PriABs than PriAEc to
ssDNA. Probably associated with this property is the
capability of PriABs to bind to bubble structures, although less
stably and with a lower affinity than to the D-loop structure.
SSBBs prevents PriABs binding to ssDNA, but not to the ssiA
sequence (this study) nor to a forked structure (data not
shown). Therefore, we propose that SSBBs protein participates
in the specific targeting of PriABs-mediated primosome
assembly to the DNA, as recently concluded for the E.coli
primosomal restart machinery (
). PriA and SSB are two
proteins highly conserved in bacteria and are two players
involved in the early steps of replication fork re-activation.
Therefore, an identical functional scaffold of replication restart
appears conserved in these microorganisms in the initial
Despite the strong similarities between the two bacterial
PriA proteins, PriABs does not substitute for PriAEc in vivo,
which shows its host specificity. We have observed that PriABs
is toxic in E.coli priA cells, but not in isogenic wild-type cells.
It is possible that PriABs competes in the mutant with the
primosomal pathways that operate in the absence of PriAEc
). Another possibility would be that production of PriABs
adds yet another defect to the priA mutant cells, which are
already affected in the metabolism of chromosomal DNA. The
strong affinity of PriABs for ssDNA might be responsible for
this toxicity. Nevertheless, the lack of toxicity of PriABs in
wild-type cells suggests that it neither competes efficiently
with the endogenous PriAEc for its regular chromosomal
substrates nor titrates other protein partners.
The host specificity of the PriA protein raises the question of
the protein content of the PriA-dependent primosome in
B.subtilis. The E.coli PriA primosomal partners are PriB, PriC,
DnaT, DnaC, DnaB (the replication fork helicase) and DnaG
(the primase). Likely counterparts of the DnaB helicase and the
DnaG primase are encoded respectively by dnaC and dnaG
(formerly dnaE) in B.subtilis (
), but no obvious homologues
of PriB, PriC and DnaT have been found in B.subtilis (27). We
have suggested that three B.subtilis proteins, DnaB, DnaD and
DnaI, initially identified as required for initiation of
chromosome replication, are primosomal proteins. Indeed, they are
required for the activity of the primosome assembly site ssiA
and are involved in the replication of pAMβ1-type plasmids
). Moreover, DnaI was shown to interact with DnaC
in a two-hybrid assay and to co-localise with DnaB in the cell
(55). Recently we isolated dnaB mutations that suppress the
phenotypes of B.subtilis priA mutants. Interestingly, the in vivo
defects of primosome assembly observed in a priA mutant
were compensated for by a dnaB mutation, in a dnaD- and
dnaI-dependent manner (
). Furthermore, we directly showed
in vitro that purified PriA, DnaD and DnaB proteins
specifically interact in this order on a forked DNA substrate,
mimicking the product of recombinational repair of a stalled
replication fork (
). Altogether, these genetic and
biochemical observations suggest that PriABs, DnaB, DnaD and DnaI
could act together to load the replication fork helicase DnaC onto
DNA during replication fork restart. Therefore, the E.coli and
B.subtilis restart primosomes have apparently diverged at the
proteins acting between the PriA initiator and the replicative
We thank D. Mazel and J. Errington for providing DNA
sequences of the priA region before publication, M. Farache
for technical help, S. Sandler and K. J. Marians for providing
the E.coli strains DM4000 and JJC18983 and the PriAEc
expression plasmid and M. -A. Petit for critical reading of the
manuscript. This work was supported, in part, by the Ministère
de l’Education Nationale, de la Recherche et de la Technologie
(Programme de Recherche Fondamentale en Microbiologie et
Maladies Infectieuses et Parasitaires) and the European
Commission (BIO4-CT98-0250). P.P. is on the CNRS staff.
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