JOURNAL OF BACTERIOLOGY, Jan. 2003, p. 340–348 Vol. 185, No.

1
0021-9193/03/$08.00⫹0 DOI: 10.1128/JB.185.1.340–348.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Forespore-Specific Expression of Bacillus subtilis yqfS, Which Encodes

Type IV Apurinic/Apyrimidinic Endonuclease, a Component of the
Base Excision Repair Pathway
Norma Urtiz-Estrada,1 José M. Salas-Pacheco,1 Ronald E. Yasbin,2 and Mario Pedraza-Reyes1*
Institute of Investigation in Experimental Biology, Faculty of Chemistry, University of Guanajuato, Guanajuato, Gto. 36060,
Mexico,1 and Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas 750832
Received 1 July 2002/Accepted 28 August 2002

Downloaded from http://jb.asm.org/ on April 7, 2015 by UCSF Library & CKM

The temporal and spatial expression of the yqfS gene of Bacillus subtilis, which encodes a type IV apurinic/
apyrimidinic endonuclease, was studied. A reporter gene fusion to the yqfS opening reading frame revealed that
this gene is not transcribed during vegetative growth but is transcribed during the last steps of the sporulation
process and is localized to the developing forespore compartment. In agreement with these results, yqfS mRNAs
were mainly detected by both Northern blotting and reverse transcription-PCR, during the last steps of
sporulation. The expression pattern of the yqfS-lacZ fusion suggested that yqfS may be an additional member
of the E␴G regulon. A primer extension product mapped the transcriptional start site of yqfS, 54 to 55 bp
upstream of translation start codon of yqfS. Such an extension product was obtained from RNA samples of
sporulating cells but not from those of vegetatively growing cells. Inspection of the nucleotide sequence lying
upstream of the in vivo-mapped transcriptional yqfS start site revealed the presence of a sequence with good
homology to promoters preceding genes of the ␴G regulon. Although yqfS expression was temporally regulated,
neither oxidative damage (after either treatment with paraquat or hydrogen peroxide) nor mitomycin C
treatment induced the transcription of this gene.

Endogenous and environmental factors such as reactive ox- hydroxyl radical-induced DNA backbone cleavage, thus con-
ygen species, UV light, and chemical carcinogens alter the tributing to spore resistance to heat and oxidizing agents (re-
chemical structure of DNA bases, producing lesions that are viewed in references 40 and 58). ␣/␤-type SASPs bind to spore
substrates for a myriad of DNA glycosylases of the base exci- DNA and are in part responsible of the strong resistance of B.
sion repair (BER) pathway (27). The apurinic/apyrimidinic subtilis spores to UV light (reviewed in references 40, 41, and
(AP) sites generated not only by the action of DNA glycosy- 58); however, these DNA-binding proteins do not confer pro-
lases but also by the spontaneous depurination and depyrim- tection to DNA against base alkylation (55).
idination of DNA (29, 30) are inherently toxic and highly The genome of B. subtilis (26) possesses genes that poten-
mutagenic and thus should be rapidly processed and elimi- tially encode ExoIII and type IV AP endonucleases, namely,
nated (31). The first catalytic event during the repair of AP exoA and yqfS, whose products show a high level of homology
sites is carried out by AP endonucleases, which cleave the to ExoIII and type IV AP endonucleases, respectively. Al-
DNA backbone immediately 5⬘ of an AP site, generating a 5⬘ though the enzymology of B. subtilis ExoA has been studied in
deoxyribose-phosphate group and a 3⬘ deoxyribose-hydroxyl detail (53), nothing has been reported regarding the mecha-
group. AP endonucleases have been classified into two fami- nisms that control its expression during growth and sporulation
lies, namely, ExoIII and type IV AP endonucleases (3, 13), and of B. subtilis.
these enzymes have been conserved across the species of the The expression of DNA repair systems in the gram-positive
three domains of life (23). spore-forming bacterium B. subtilis has been shown to be dif-
Dormant spores of Bacillus subtilis are more resistant than ferentially regulated during growth and differentiation (4, 11,
their vegetatively growing counterparts to several chemical 32, 34), as well as during spore germination and outgrowth
substances, including acids, bases, alkylating agents, and oxi- (54). DNA lesions acquired during unpredictable periods of B.
dizing agents (reviewed in references 40, 41, and 58). The subtilis spores dormancy must be necessarily corrected during
existence of core coats, the low permeability of spores to hy- germination by spore-specific expressed DNA repair systems
drophilic compounds, and the protection of spore DNA from (reviewed in references 40 and 58). The best example studied
damage by its saturation with ␣/␤-type small acid-soluble pro- thus far is the correction of the UV-C induced spore photo-
teins (SASPs) account for this resistance (reviewed in refer- product (5-thyminil-5,6-dihydrothymine) through both the spe-
ences 40, 56, and 58). It has been demonstrated that ␣/␤-type cific spore photoproduct lyase protein (SplB) and the general
SASPs slow DNA depurination-depyrimidination, as well as excision-repair system (UVR) (reviewed in references 40 and
41). However, during unpredicted periods of spore dormancy
B. subtilis spores could potentially accumulate, in addition to
* Corresponding author. Mailing address: Institute of Investigation spore photoproduct (SP), different types of DNA lesions, such
in Experimental Biology, Faculty of Chemistry, University of Guana-
juato, P.O. Box 187, Guanajuato, Gto. 36060, Mexico. Phone: (473)
as strand brakes, cyclobutane pyrimidine dimers (CPDs),
73-2-00-06, x8161. Fax: (473) 73-2-00-06, x8153. E-mail: pedrama chemically altered bases, and AP sites that could affect essen-
@quijote.ugto.mx. tial functions such as transcription and replication during ger-

340
VOL. 185, 2003 B. SUBTILIS TYPE IV AP ENDONUCLEASE 341

TABLE 1. Strains and plasmids used in this study

Strain or plasmid Genotype and/or phenotypea Source (reference)

Strains
B. subtilis
168 trpC2 Laboratory stock
WN118 sigG⌬1 trpC2 Wayne Nicholson
PERM317 trpC2 yqfS-lacZ; Cmr This study
PERM336 sigG⌬1 trpC2 yqfS-lacZ This study
YB3000 metB5 trpC2 xin- 1 sigB amyE (deleted for sp␤) pCCR202 (recA-lacZ at amyE); Cmr R. E. Yasbin

E. coli
SURE e14⫺(McrA⫺) ⌬(mcrCB-hsdSMR-mrr)171 endA1 supE44 thi-1 gyrA96 relA1 lac recB Stratagene
recJ sbcC umuC::Tn5 (Kanr) uvrC [F⬘ proAB laclq lacZ⌬M15 Tn10 (Tetr)]
PERM162 E. coli SURE, pUC18; Ampr Tcr This study

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PERM253 E. coli SURE, pPERM253; Ampr Tcr This study
XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac/F⬘ proAB lacIqZ⌬ M15 Tn10 (Tetr) Stratagene
PERM267 E. coli XL1-Blue, pPERM253; Ampr This study
XL10-Gold Kan Tetr ⌬(mcrA) 183, ⌬(mcrCB-hsdSMR-mrr); 173 endA1 sup E44 thi-1 recA1 gyrA96 Stratagene
relA1 lacHte [F⬘ proAB lacIqZ⌬M15 Tn10 (Tetr) Tn5 (Kanr) Amy]
PERM348 E. coli XL-Gold, pPERM348, Ampr This study

Plasmids
pJF751 Integrational lacZ fusion vector; Cmr W. Nicholson (17)
pUC18 Multisite E. coli cloning vector Laboratory stock (63)
pPERM253 yqfS gene cloned in pUC18 This study
pPERM267 514-pb EcoRI/NaeI fragment of yqfS from pPERM253 cloned in pJF751 This study
pPERM348 yqfS ORF cloned into the BamHI site of PQE-30 This study
a
Cmr, chloramphenicol resistant; Ampr, ampicillin resistant; Tcr, tetracycline resistant.

mination (40). Although the expression of splB in the forespore commercial ion-exchange columns according to the instructions of the supplier
compartment by ␴G RNA polymerase has been widely sub- (Qiagen, Inc., Valencia, Calif.). Nucleic acid sequencing by dideoxynucleotide
chain termination (50) was performed with the Thermo Sequenase radiolabeled
stantiated (44, 45), few data exist in the literature concerning terminator cycle sequencing kit (U.S. Biochemical Corporation, Cleveland,
the expression of other specific or general DNA repair systems Ohio). Sequencing products were analyzed by autoradiography after electro-
in the forespore compartment. phoresis through a 6% polyacrylamide sequencing gel. Alternatively, DNA plas-
As mentioned above, in the genome of B. subtilis exists an mids purified through Qiagen columns were processed for sequencing in a
open reading frame (ORF), yqfS, whose predicted product Perkin-Elmer (Norwalk, Conn.) model 377A automated DNA sequencer.
Cloning of yqfS and construction and integration of a yqfS-lacZ gene fusion.
shows 53% homology with the type IV AP endonuclease of The complete yqfS gene was amplified by PCR with genomic DNA from B.
Escherichia coli. We describe here the expression of the cloned subtilis 168 as a template and the oligonucleotide primers 5⬘-GGGAATTCGC
yqfS gene of B. subtilis from an IPTG (isopropyl-␤-D-thioga- CGAAGAAGGTTAAGCC-3⬘ (forward) and 5⬘-CGGGATCCGGCCGTTGAA
lactopyranoside)-inducible promoter in E. coli. Our results GTAGCGAACC-3⬘ (reverse). The primers were designed to insert EcoRI and
demonstrate that a His6-YqfS purified enzyme is able to pro- BamHI sites (underlined). Amplification was performed on 0.1 ␮g of chromo-
somal DNA by using an MJ Research (Watertown, Mass.) Minicycler with Vent
cess the cleavage of abasic sites in the DNA. In addition, our DNA polymerase (New England Biolabs, Beverly, Mass.) according to the man-
results demonstrated that the expression of yqfS is forespore ufacturer’s recommendations. The 1,181-bp PCR fragment extending from 110
specific but was not induced by the stress imposed by super- bp upstream of the yqfS start codon through 157 bp downstream of the yqfS stop
oxide radicals, by hydrogen peroxide, or by the DNA-damaging codon was digested with SmaI and BamHI and ligated into pUC18 to generate
agent mitomycin C. pPERM253. pPERM253 was replicated in E. coli XL1-Blue, and the cloned yqfS
gene was sequenced on both strands.
Construction of an in-frame translational yqfS-lacZ fusion was performed in
MATERIALS AND METHODS the integrative plasmid pJF751 (17) by inserting a 472-bp EcoRI-NaeI fragment
Bacterial strains, plasmids, and growth conditions. B. subtilis and E. coli from plamid pPERM253 into pJF751 previously digested with EcoRI and SmaI.
strains used in the present study are shown in Table 1. Plasmids used in this work The resulting construction, containing the yqfS-lacZ fusion and designated
are listed in Table 1. Media used were Difco sporulation medium (DSM) (52) pPERM317, was propagated into E. coli XL1-Blue. Plasmid pPERM317 was
and Luria-Bertani (LB) medium (38). When appropriate, antibiotics were added introduced by transformation into competent cells of B. subtilis 168, and trans-
to the medium at the following final concentrations: chloramphenicol, 3 ␮g/ml; formants were selected on solid DSM containing chloramphenicol.
ampicillin, 50 ␮g/ml; and kanamycin, 10 ␮g/ml. Liquid cultures were shaken at Purification of His6-YqfS and substrates for AP endonuclease activity. E. coli
250 rpm at 37°C. Cultures on solid medium were grown at 37°C. The optical PERM348 containing plasmid pPERM348 (Table 1) was grown in 50 ml of LB
density (OD) of liquid cultures was monitored with a Pharmacia Ultrospec 2000 medium, supplemented with ampicillin (100 ␮g/ml), at 37 oC to an OD at 600 nm
spectrophotometer set at 600 nm. (OD600) of 0.5. Expression of yqfS was induced during 4 h at 37°C by the addition
Genetic and molecular biology techniques. Preparation of competent E. coli or of IPTG to 0.5 mM. Cells were collected by centrifugation and washed two times
B. subtilis cells and their transformation with DNA was performed as described with 10 ml of 50 mM Tris-HCl (pH 7.5)–300 mM NaCl (buffer A). The cells were
elsewhere (5, 49). Extraction of chromosomal DNA from B. subtilis was carried disrupted in 10 ml of the same buffer containing lysozyme (10 mg/ml) for 30 min
out according to the protocol of Cutting and Vander Horn (12). Small-scale at 37°C. The cell homogenate was subjected to centrifugation to eliminate un-
preparation of plasmid DNA from E. coli cells, enzymatic manipulations, and disrupted cells and cell debris, and the supernatant was applied to a 5-ml nickel-
agarose gel electrophoresis were performed by standard techniques (49). Large- nitrilotriacetic acid-agarose column previously equilibrated with buffer A. The
scale preparation and purification of plasmid DNA was accomplished by using column was washed with 50 ml of buffer A containing 10 mM imidazole plus 50
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FIG. 1. yqfS region of the B. subtilis chromosome and DNA sequences lying upstream of the yqfS ORF. (A) Genetic organization of the yqfS
locus between indicated coordinates of the B. subtilis chromosome (filled box). Dashed lines above the ORFs (arrows) show the DNA fragments
cloned into the indicated plasmids. Downstream of yqfU ORF a putative transcriptional terminator is shown (stem-loop structure). (B) Sequence
of the intergenic region between yqfR and yqfS. The in vivo-mapped transcriptional start site of yqfS is indicated by an asterisk immediately
downstream of the ⫺10 and ⫺35 sequences that might function as a promoter for RNA polymerase-␴G. RBS (putative ribosome-binding site).

ml of buffer A containing 20 mM imidazole, and the protein bound to the resin tive and sporulating cells of B. subtilis PERM317. In order to obtain the maxi-
was eluted with 15 ml of buffer A containing 100 mM imidazole; 2-ml fractions mum amount of yqfS transcripts during sporulation, we monitored the expression
were collected during this last step. Aliquots (15 ␮l) of the cell homogenate, the of ␤-galactosidase activity directed by the yqfS-lacZ fusion in this strain. The total
flowthrough, and the bound fractions were analyzed by sodium dodecyl sulfate- RNA (40 ␮g from each sample) was hybridized with the 20-mer oligonucleotide
polyacrylamide gel electrophoresis. 5⬘-CGGCGCGTATTTTGCGGTGC-3⬘, which was complementary to the yqfS
Two types of substrates were prepared to assay AP endonuclease activity of mRNA from nucleotides 106 to 124 downstream from the putative yqfS trans-
His6-YqfS, namely, pBluescript (Stratagene), which was partially depurinated lational start codon. The oligonucleotide was labeled on its 5⬘ end with
after a previously described protocol (28) and a 5⬘-end-radiolabeled double- [␥-32P]ATP and T4 polynucleotide kinase. The primer was extended with Molo-
stranded 19-mer nucleotide containing a single abasic site (20). ney murine leukemia virus reverse transcriptase, and the extended products were
The endonuclease activity of His6-YqfS against pBluescript containing AP separated by electrophoresis through a 6% polyacrylamide DNA sequencing gel.
sites (AP-pB) was determined in a mixture reaction of 25 ␮l containing 600 ng of The position of the extended products was determined by running a sequencing
purified His6-YqfS and 100 ng of substrate in 50 mM Tris-HCl (pH 7.5) con- reaction generated with the same 20-base primer and a 1,978-bp PCR product
taining 1 mM dithiothreitol. The reactions were incubated at 37°C for 30 min and (PCR RS) extending from 247 bp upstream of the yqfR start codon to 416 bp
analyzed by electrophoresis on a 1% agarose gel stained with ethidium bromide. downstream of the start codon of yqfS (Fig. 1).
Endonuclease activity against the double-stranded radiolabeled 19-mer con- RT-PCR experiments. Total RNA from vegetative or sporulating B. subtilis
taining a single AP site was performed in a total volume of 15 ␮l containing 50 168 cells, grown in DSM, was isolated by using the TRI reagent (Molecular
mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, and 500 nmol of unlabeled and 10 Research Center, Inc.). Reverse transcription-PCRs (RT-PCRs) were performed
nmol of double-stranded radiolabeled 19-mer containing a single AP site. Dif- with the RNA samples and a Master Amp RT-PCR kit (Epicentre Technologies)
ferent amounts of His6-YqfS were added to the mixture reactions and incubated according to the instructions of the provider. The primers used for RT-PCRs
for 30 min at 37°C. The reactions were separated on a 20% denaturing acryl- were 5⬘-CCTGTTGCTGAGAATAGGC-3⬘ (forward) and 5⬘-CGGCGCGTAT
amide gel and then subjected to autoradiography. TTTGCGGTGG-3⬘ (reverse) to generate a 132-bp RT-PCR product extending
Cell growth and enzymatic assays. B. subtilis strains carrying the yqfS-lacZ from 4 bp upstream from the start codon of yqfS to 128 bp downstream of this
fusion were grown and allowed to sporulate in liquid DSM. Samples of 1.5 ml point (Fig. 1). As a control, in each experiment, the absence of chromosomal
were collected during vegetative growth and throughout sporulation. Cells were DNA in the RNA samples was assessed by mounting PCRs with Vent DNA
washed with 0.1 M Tris-HCl (pH 7.5) and processed for determination of ␤-ga- polymerase (New England Biolabs) and the set of primers described above.
lactosidase (42) and glucose dehydrogenase (GDH) activities (19, 42). The ␤-ga-
lactosidase activities were determined in cell extracts obtained from mother cells
and forespore fractions prepared according to a previously described protocol
(36, 44). RESULTS
Northern blot and primer extension experiments. The total RNA for both
Northern blotting experiments and mapping of the 5⬘end of yqfS was isolated as Cloning of yqfS. The existence of a type IV AP endonuclease
previously described (35). Northern blots were performed with RNA samples in the genome of B. subtilis was investigated by using the
isolated from strains B. subtilis 168 and WN118 (sigG mutant). RNA samples (20 primary structure of E. coli Nfo (51) as a query to search
␮g) were separated by electrophoresis through 1% agarose-formamide gel and against the database of National Center for Biotechnology
transferred to a high-bound nylon membrane. The membrane containing the
transferred RNA was hybridized at 70°C with a 1,181-pb EcoRI-BamHI frag-
Information with a Gapped BLAST program (2). As described
ment from pPERM253 containing the entire yqfS sequence. The probe was in Materials and Methods, this approach was used to retrieve
labeled by random priming with [␣-32P]dCTP by using the Rediprime II DNA a gene termed yqfS from the genome of B. subtilis (26). Anal-
labeling system according to the instructions of the provider (Amersham Bio- ysis of the yqfS primary structure revealed an ORF of 891 bp
sciences, Buckinghamshire, England). Detection of hybrids was performed by
autoradiography exposing the membrane to Kodak X-Omat films.
with enough information for the synthesis of a predicted pro-
The 5⬘ end of yqfS was mapped by primer extension (37) of yqfS transcripts tein of 31 kDa. Amino acid alignments showed that YqfS
produced during sporulation. To this end, total RNA was isolated from vegeta- possesses homologies of 53, 52, and 32% with E. coli Nfo (51),
VOL. 185, 2003 B. SUBTILIS TYPE IV AP ENDONUCLEASE 343

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FIG. 2. Endonuclease activity of His6-YqfS against a plasmid con-
taining AP sites. Aliquots (600 ng) of His6-YqfS were incubated with
100 ng of either untreated (U-pB [lane 4]) or AP-containing sites
(AP-pB [lane 3]) of pBluescript. Lane 1, AP sites-containing plasmid
incubated with 50 mM Tris-HCl (pH 7.5)–300 mM NaCl; lane 2,
untreated plasmid incubated with 50 mM Tris-HCl (pH 7.5)–300 mM
NaCl. The reactions were incubated at 37°C for 30 min and then
analyzed by electrophoresis on a 1% agarose gel stained with ethidium
bromide.

Saccharomyces cerevisiae Apn1 (46), and Thermotoga maritima

endonuclease IV (20), respectively. FIG. 3. Endonuclease activity of His6-YqfS against a double-
stranded 19-mer containing a single AP site. (A) A total of 510 nmol
Purification and enzymatic activity of YqfS. The His6-YqfS of 5⬘-end-radiolabeled double-stranded 19-mer nucleotide containing
protein synthesized in E. coli was purified to homogeneity by a single AP site was incubated for 30 min at 37°C with different
metal chelate affinity chromatography, yielding a 36-kDa pro- concentrations of His6-YqfS. The reactions were separated on a 20%
tein (data not shown). denaturing acrylamide gel and then subjected to autoradiography.
To corroborate the predicted AP endonuclease activity of Lane 1, no enzyme; lanes 2 to 6, 0.3, 0.6, 1.2, 2.4, and 3.6 ␮g of
His6-YqfS, respectively; lane 7, 2 U of E. coli Nfo. Radioactively
YqfS, two enzymatic assays were performed. First, the His6- labeled cleaved (C) and uncleaved (U) strands are as indicated.
YqfS pure enzyme was incubated with a partially depurinated (B) Densitometry of the experiment shown in panel A; the percentage
plasmid DNA as a substrate (AP-pB). The results presented in of uncleaved substrate was plotted as a function of the amount of
Fig. 2 reveal the conversion of the closed covalently circular His6-YqfS added to the reaction.
depurinated plasmid (CCC) to the open circular form (OC)
due to single-strand breaks performed by the His6-YqfS puri-
fied protein (lane 3). As shown in Fig. 2 (lane 4), the nonde- time that the product encoded by the yqfS gene possesses
purinated plasmid (U-pB) was not a substrate for the His- activity of AP endonuclease, a result in agreement with its high
tagged YqfS protein. Controls shown in Fig. 2 revealed that structural similarity to the family IV AP endonucleases.
neither the untreated nor the depurinated plasmid were con- Expression of a yqfS during growth and sporulation. The
verted to the OC form in the absence of the His6-YqfS protein strain B. subtilis PERM317 harboring a single copy of the
(lanes 1 to 2). Second, a 5⬘-end radiolabeled double-stranded yqfS-lacZ fusion was grown in DSM to induce sporulation.
19-mer nucleotide containing a single AP site was used as a Determination of yqfS-directed ␤-galactosidase activity during
substrate for the YqfS pure protein. Essentially, different growth and sporulation stages revealed a temporal pattern of
amounts of the His6-tagged protein were incubated with 510 expression. Although no ␤-galactosidase activity was detected
nM of this AP substrate. The products of the reaction analyzed during vegetative growth, Fig. 4 reveals that enzymatic activity
on a denaturing polyacrylamide gel revealed that the endonu- was detectable after T0, reached a maximum during T6 and T7,
cleolytic activity of YqfS at the AP site was dependent on the and then decreased. The expression pattern of the reporter
concentration of the enzyme used (Fig. 3A, lanes 2 to 6). To gene (Fig. 4) was similar to that observed for genes whose
better evaluate this conclusion, these results were analyzed by expression occurs during the last steps of sporulation in the
densitometry, thereby corroborating that cleavage of the AP forespore compartment, such as the operon splA-splB (44), gdh
substrate by His6-YqfS is concentration dependent (Fig. 3B). (39), and ssp (35, 36) genes. To further investigate this obser-
Although the radiolabeled 20-bp-mer was also cleaved by E. vation, two approaches were followed. First, cell fractioning
coli Nfo (Fig. 3A, lane 7), it was observed that a fraction of the experiments were performed to investigate whether the expres-
substrate was partially degraded (Fig. 3A, lane 1). The results sion of the reporter gene occurred inside of the spore. The
presented in Fig. 3A (lane 7) also revealed that another frac- results of Fig. 4 show that ␤-galactosidase activity started to
tion of the radiolabeled substrate was inaccessible to the en- accumulate inside of the forespores from sporulation stage T5
zyme; such a fraction most probably corresponded to nonde- and continued to accumulate until at least stage T9.
purinated compound. These results demonstrate for the first The cell extracts used to determine ␤-galactosidase activity
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FIG. 5. Northern blot (A) and RT-PCR analysis (B) of yqfS tran-
scription during vegetative growth and sporulation. (A) B. subtilis 168
FIG. 4. Expression of a yqfS-lacZ translational fusion during was induced to sporulate in liquid DSM. Total RNA was isolated (35)
growth and sporulation of B. subtilis. B. subtilis PERM317 was grown during the times (in hours) indicated (T0 ⫽ end of exponential
to sporulation in liquid DSM (■). Samples were collected at different growth). Then, 20-␮g samples were separated on agarose-formalde-
times and treated with lysozyme, and the extracts were assayed for hyde gels (lower panel) and transferred to nylon membranes. The
either ␤-galactosidase (⽧) or GDH (F) activity. The ␤-galactosidase membrane was hybridized with a 32P-labeled 1,181-bp fragment en-
activity inside of the forespore lysozyme-resistant fraction (Œ) was compassing the entire yqfS sequence as described in Materials and
assayed as described in Materials and Methods. Methods. (B) RNA samples (1 ␮g) isolated from a B. subtilis 168 DSM
culture at the times indicated (in hours) were processed for RT-PCR
analysis as described in Materials and Methods. The arrowhead shows
the size of the expected RT-PCR product.
were also assayed for GDH activity, an enzyme encoded by the
stage III, forespore-specific gdh gene (19, 39). The results
shown in Fig. 4 revealed that the expression patterns of the stationary-growth phases (data not shown). Consistent with
yqfS-lacZ fusion and the GDH activity followed essentially this result, the levels of GDH in this strain were almost zero
identical kinetics, strongly indicating that yqfS gene expression (data not shown).
is activated in the forespore compartment during the last steps In a second approach, Northern blot experiments were per-
of sporulation. formed with RNA isolated from vegetative and stationary cells
To further support this contention, Northern blot experi- of B. subtilis sigG⌬1 grown in liquid DSM. The results shown in
ments were performed with total RNA isolated from cells of Fig. 6A revealed the lack of yqfS mRNAs in this sigG mutant
strain B. subtilis PERM168 collected before and after the onset genetic background, since no hybridization signal was detected
of sporulation. The results (Fig. 5A) indicated that yqfS mRNA during both exponential-growth-phase and stationary-growth-
appeared as a 2.3-kb band during sporulation stages T5 phase cells. Such a result was also confirmed by RT-PCR
through T9, observing a major hybridization signal at T7. As experiments, which failed to amplify the 132-bp yqfS fragment
shown in Fig. 5A, no signal was detected in the blot with RNA from RNA samples isolated before and after the onset of
isolated from cells growing exponentially, supporting the con- sporulation (Fig. 6B). Taken together, these results are con-
clusion that yqfS expression is sporulation specific. Moreover, sistent with yqfS expression being dependent on ␴G RNA poly-
RT-PCR experiments resulted in the major amplification of a merase.
yqfS product when total RNA isolated from sporulating cells Mapping the transcriptional start site of yqfS. The genetic
was used as a template. Figure 5B shows that the RT-PCR organization of the yqfS locus reveals that this gene is flanked
product of yqfS (132 bp) was more abundant with RNA sam- upstream by yqfR, which encodes a putative RNA helicase, and
ples of the step T7 of sporulation. downstream by yqfU, which encodes a protein of unknown
␴G dependence of yqfS expression. The expression of fore- function (Fig. 1). The existence of only one potential transcrip-
spore specific genes in B. subtilis is carried out through the tional terminator until the end of yqfU suggests that the three
sequential action of two temporally expressed RNA poly- genes could be cotranscribed as a polycistronic message. To
merases containing either ␴F or ␴G factors (21, 43). However, investigate this possibility, primer extension analysis was per-
as shown above, the expression pattern of the yqfS-lacZ fusion formed to map the 5⬘ ends of the mRNAs originating from
was very similar to the ␴G-dependent gdh gene, suggesting that upstream from the yqfS coding sequence. Experiments were
yqfS is under the control of ␴G-containing RNA polymerase. carried out with total RNA isolated from B. subtilis PERM317
This notion was directly tested by two different approaches. harboring the yqfS-lacZ fusion. Cells used to isolate RNA were
First, the yqfS-lacZ fusion was introduced by transformation harvested during both vegetative growth and the T7 sporula-
into competent cells of B. subtilis WN126 harboring a deletion tion stage, the time of maximum expression of the yqfS-lacZ
of the ␴G gene, an spo mutant in which sporulation is arrested fusion. The results shown in Fig. 7 (lane 2) revealed the syn-
during stage III (24, 60). The resulting strain, B. subtilis thesis of a major extension product located 54 to 55 bp up-
PERM336, grown in DSM expressed very low levels of yqfS- stream of translation start codon of yqfS. Such an extension
directed ␤-galactosidase activity during both vegetative- and product was obtained only in experiments performed with
VOL. 185, 2003 B. SUBTILIS TYPE IV AP ENDONUCLEASE 345

FIG. 8. Comparison of the consensus E␴G (19) promoter sequence

(top line) with the putative promoter sequence lying upstream of yqfS
(bottom line). Absolutely conserved (boldface) or highly conserved
(underlined) bases in E␴G-type promoters (21, 43). The position of the
mapped transcriptional start site of yqfS is indicated with an asterisk.

absolutely conserved bases present on sigG promoters (Fig. 8).

On the other hand, the ⫺10 region conserved three of the four

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absolutely conserved residues observed in such ␴G promoters.
However, it was found that the ⫺10 and ⫺35 regions were
FIG. 6. Northern blot (A) and RT-PCR analysis (B) of yqfS tran- separated by 16 bp instead of the reported 17 to 18 bp for the
scription during vegetative growth and sporulation of B. subtilis sigG⌬1 ␴G consensus sequence (Fig. 8).
(strain WN118). (A) B. subtilis WN118 was grown in liquid DSM. Total Induction of the yqfS-lacZ fusion by oxidative stress or dur-
RNA was isolated (35) during the times indicated (in hours). Samples
ing the SOS response. In E. coli, the expression of the type IV
(20 ␮g) were separated on agarose-formaldehyde gels (lower panel)
and transferred to nylon membranes. The membrane was hybridized AP endonuclease nfo gene is induced by generators of super-
with a 32P-labeled 1,181-bp fragment encompassing the entire yqfS oxide radicals, such as paraquat (8). On the other hand, B.
sequence as described in Materials and Methods. (B) RNA samples (1 subtilis responds to H2O2 stress displaying an adaptive re-
␮g) isolated at the times indicated (in hours) from a B. subtilis sigG⌬1 sponse that induces the expression of genes such as katA (cata-
DSM culture were processed for RT-PCR analysis as described in
Materials and Methods. For the wild type (WT), the RNA was isolated lase), ahpCF (alkyl hydroperoxide reductase), mrgA, and the
from B. subtilis 168 (Fig. 5); FW was obtained with the forward primer hemA operon (1, 4, 7, 9, 10, 14). We therefore investigated
in the absence of RNA, and RV was obtained with the reverse primer whether the yqfS gene in B. subtilis is also induced by the
in the absence of RNA. The arrowhead shows the size of the expected oxidative stress imposed by either superoxide radicals or hy-
RT-PCR product.
drogen peroxide. To this end, the strain B. subtilis PERM317
containing the yqfS-lacZ fusion, integrated into the yqfS locus,
was grown in LB medium to the mid-exponential phase and
RNA isolated from sporulating cells but not with RNA of treated with paraquat (10 ␮M) or hydrogen peroxide (200
vegetatively growing cells (Fig. 7, lane 1). Inspection of the ␮M). The results (Fig. 9A) revealed that, at the concentrations
nucleotide sequences lying upstream of the in vivo mapped
transcriptional yqfS start site revealed the existence of se-
quences with good homology to promoters preceding genes of
the ␴G regulon (Fig. 8) (21, 43). A higher level of homology
was found in the ⫺35 region, which possessed three of the four

FIG. 9. Lack of induction of a yqfS-lacZ fusion by paraquat, H2O2,

or mitomycin. B. subtilis PERM317 was grown to an OD600 of 0.5 in
either minimal Spizizen medium (A) or LB medium (B). The culture
FIG. 7. Primer extension analysis for mapping the transcriptional made in minimal Spizizen medium was divided into three subcultures;
start site of yqfS. Total RNA was isolated (34) from either vegetative one (labeled “0”) was left untreated, and the other two were treated
(lane 1) or sporulating (stage T7; lane 2) B. subtilis PERM317 cells with either paraquat (PQ; 10 ␮M) or H2O2 (200 ␮M). The LB culture
grown in DSM. Primer extension was performed as described in Ma- was treated in the same manner except that mitomycin C (MC; 0.5
terials and Methods. The asterisk indicates the position of the primer ␮g/ml) was added to the culture. (C) B. subtilis YB3000 was grown in
extension product in the DNA sequence lying upstream of yqfS (see LB medium to an OD600 of 0.5; at this point, the culture was equally
Fig. 1). The 5⬘ end of the yqfS transcript was determined by running a divided, and mitomycin C (0.5 ␮g/ml) was added to one of the sub-
DNA sequencing ladder generated with the same primer (lanes G, A, cultures. In all cases, the ␤-galactosidase activity was determined with
T, and C) and was labeled with an arrowhead. cell samples collected 2 h after the addition of the inducers.
346 URTIZ-ESTRADA ET AL. J. BACTERIOL.

tested, neither paraquat nor H2O2 was capable of inducing the transcription of yqfS is carried out by RNA polymerase con-
expression of the yqfS-lacZ fusion. taining the ␴G factor (Fig. 4). However, gene expression inside
Several B. subtilis genes involved in DNA repair, such as uvr of the forespore occurs by the sequential action of two RNA
components and recA, have been shown to be inducible not polymerases containing either the ␴F or ␴G factors (21, 25).
only by DNA damage but also by the physiological state of Therefore, we could not rule out a possible transcription of
competence (32, 34, 48). These genes (din) are part of a global yqfS by RNA polymerase ␴F. This point was addressed by
response which in B. subtilis is called the SOS response (33). In measuring the levels of expression of the yqfS-lacZ fusion in-
order to determine whether the type IV AP-endonuclease troduced into a B. subtilis strain lacking the sigG gene (Table
gene of B. subtilis is a component of the B. subtilis SOS regu- 1). The results showed that yqfS-directed ␤-galactosidase ac-
lon, the strain containing the yqfS-lacZ fusion was grown to tivity is almost null in this genetic background, as is the syn-
exponential phase and then treated with mitomycin C to a final thesis of GDH activity (data not shown). In agreement with
concentration of 0.5 ␮g/ml. As shown in Fig. 9B, mitomycin C this observation, both Northern blot and RT-PCR experiments
induced the ␤-galactosidase levels of the strain B. subtilis performed with total RNA isolated during vegetative and sta-

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PERM317 only 1.2 times above the levels expressed by the tionary growth of the strain B. subtilis sigG⌬1 demonstrated the
untreated control. In contrast with this result, when B. subtilis absence of yqfS messengers in this mutant strain (Fig. 6).
YB3000 containing a recA-lacZ fusion was treated with mito- Taken collectively, these results strongly suggest that yqfS ex-
mycin (Fig. 9C), the levels of ␤-galactosidase activity increased pression occurs inside of the spore by the action of ␴G-con-
35 times. taining RNA polymerase.
Forespore-specific expressed genes such as sspA-E, splA-
DISCUSSION splB, gdh, ger, and spoVA, among others, are representative of
the ␴G regulon (15, 16, 19, 21, 25, 44, 45, 57). Experimental
B. subtilis has been studied extensively as a paradigm for evidence has demonstrated that these genes possess specific
bacterial differentiation and development. Spores produced by promoters that are exclusively transcribed by ␴G containing
this organism prevent or dramatically slow the DNA damage RNA polymerase (15, 39, 43, 44, 47). The results described
inflicted by oxidative stress, UV light, heat and desiccation above suggest that yqfS might be a new member of this regulon.
(reviewed in references 40 and 58). However, during long This conclusion was strongly supported by the in vivo mapping
periods of dormancy spores accumulate potentially lethal and of the transcriptional start site of yqfS (Fig. 7). A major exten-
mutagenic DNA damage such as SP, strand brakes, CPDs, sion product initiating 54 to 55 bp upstream of the putative
chemically altered bases, and AP sites that could affect tran- yqfS start codon was amplified from RNA samples isolated
scription and replication during germination (40, 56). There- from sporulating but not from vegetatively growing cells (Fig.
fore, it is of interest to determine how the many DNA repair 7). Inspection of the sequences preceding the yqfS transcrip-
systems present are regulated by B. subtilis, especially in rela- tional start site revealed the existence of a promoter with
tion to the sporulation and germination processes. homology to the consensus sequence of ␴G promoters (21, 43).
Thus, the yqfS ORF was cloned, and the product of this gene Although the ⫺10 region of the putative yqfS promoter shows
was isolated and tested for its enzymatic activity. The results a low level of homology, the ⫺35 region almost perfectly
presented in Fig. 2 and 3 clearly indicate that this protein has matched the consensus of ␴G promoters (Fig. 8). One possible
AP endonuclease activity. Having established the nature of the problem with the designation of this putative ␴G promoter is
product of the yqfS gene, we wanted to determine the mech- the spacing between the ⫺35 and ⫺10 regions. However, as
anism(s) that control the expression of this gene. Our data mentioned above, our data support the hypothesis that the yqfS
demonstrate that there is temporal and spatial expression of gene is transcribed by a ␴G-containing RNA polymerase.
the yqfS gene. Specifically, ␤-galactosidase activity for a yqfS- The yqfS region in the B. subtilis chromosome shows the
LacZ reveals that this gene is not apparently transcribed dur- existence of a set of three genes located in the same orienta-
ing vegetative growth but is transcribed during stages of the tion, in the following order: yqfR, yqfS, and yqfU (Fig. 1). The
sporulation process (Fig. 4). Northern blot and RT-PCR ex- lack of putative transcriptional terminators downstream of
periments (Fig. 5) confirmed a major abundance of yqfS mes- yqfR and yqfS suggests that the three genes are transcribed as
sengers during stages of the sporulation process of the strain B. a polycistronic unit. However, the primer extension experi-
subtilis PERM317. These results suggested that yqfS expression ments described above, together with the identification of a
is temporally activated and confined to the forespore compart- 2.3-kb yqfS messenger, indicate that yqfS is cotranscribed with
ment in accordance with a pattern similar to that described for yqfU as a bicistronic mRNA from the putative yqfS promoter
stage III, forespore-specific genes (57). This suggestion was just described.
further supported not only by cell fractionation experiments, Expression of the two major AP endonucleases is differen-
which demonstrated that yqfS expression occurs inside of the tially regulated in E. coli. Whereas exoIII is constitutively ex-
spore, but also by the observation that the kinetics of GDH pressed, the nfo gene is inducible by oxidative stress. Chemical
synthesis, a stage III, forespore-specific marker, are indistin- compounds such as paraquat and menadione, which generate
guishable from those observed for the yqfS-lacZ fusion (Fig. 4). superoxide radicals, induce a 10- to 20-fold increase in the level
These results strongly support the idea that the synthesis of the of Nfo (8). The lack of induction in the levels of expression of
YqfS protein occurs during the last stages of the sporulation the yqfS-lacZ fusion after the treatment of B. subtilis
process and is packaged in the spore. PERM317 with paraquat (Fig. 9) revealed that in B. subtilis the
The forespore-specific expression of the yqfS-lacZ fusion yqfS gene is not regulated by the oxidative stress imposed by
during the last steps of B. subtilis sporulation suggested that the superoxide radicals.
VOL. 185, 2003 B. SUBTILIS TYPE IV AP ENDONUCLEASE 347

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