Forespore-specific expression of Bacillus subtilis yqfS, which encodes type IV apurinic/apyrimidinic endonuclease, a component of the base excision repair pathway

Abstract

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 Esigma(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 sigma(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.

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-σ^G. RBS (putative ribosome-binding site).

FIG. 2.

Endonuclease activity of His₆-YqfS against a plasmid containing 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.

FIG. 3.

Endonuclease activity of His₆-YqfS against a double-stranded 19-mer containing a single AP site. (A) A total of 510 nmol of 5′-end-radiolabeled double-stranded 19-mer nucleotide containing a single AP site was incubated for 30 min at 37°C with different concentrations of His₆-YqfS. The reactions were separated on a 20% denaturing acrylamide gel and then subjected to autoradiography. Lane 1, no enzyme; lanes 2 to 6, 0.3, 0.6, 1.2, 2.4, and 3.6 μg of His₆-YqfS, respectively; lane 7, 2 U of E. coli Nfo. Radioactively labeled cleaved (C) and uncleaved (U) strands are as indicated. (B) Densitometry of the experiment shown in panel A; the percentage of uncleaved substrate was plotted as a function of the amount of His₆-YqfS added to the reaction.

FIG. 4.

Expression of a yqfS-lacZ translational fusion during growth and sporulation of B. subtilis. B. subtilis PERM317 was grown to sporulation in liquid DSM (▪). Samples were collected at different times and treated with lysozyme, and the extracts were assayed for either β-galactosidase (♦) or GDH (•) activity. The β-galactosidase activity inside of the forespore lysozyme-resistant fraction (▴) was assayed as described in Materials and Methods.

FIG. 5.

Northern blot (A) and RT-PCR analysis (B) of yqfS transcription during vegetative growth and sporulation. (A) B. subtilis 168 was induced to sporulate in liquid DSM. Total RNA was isolated (35) during the times (in hours) indicated (T₀ = end of exponential growth). Then, 20-μg samples were separated on agarose-formaldehyde gels (lower panel) and transferred to nylon membranes. The membrane was hybridized with a ³²P-labeled 1,181-bp fragment encompassing the entire yqfS sequence as described in Materials and Methods. (B) RNA samples (1 μg) 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.

FIG. 6.

Northern blot (A) and RT-PCR analysis (B) of yqfS transcription during vegetative growth and sporulation of B. subtilis sigGΔ1 (strain WN118). (A) B. subtilis WN118 was grown in liquid DSM. Total RNA was isolated (35) during the times indicated (in hours). Samples (20 μg) were separated on agarose-formaldehyde gels (lower panel) and transferred to nylon membranes. The membrane was hybridized with a ³²P-labeled 1,181-bp fragment encompassing the entire yqfS sequence as described in Materials and Methods. (B) RNA samples (1 μg) isolated at the times indicated (in hours) from a B. subtilis sigGΔ1 DSM culture were processed for RT-PCR analysis as described in Materials and Methods. For the wild type (WT), the RNA was isolated from B. subtilis 168 (Fig. 5); FW was obtained with the forward primer in the absence of RNA, and RV was obtained with the reverse primer in the absence of RNA. The arrowhead shows the size of the expected RT-PCR product.

FIG. 7.

Primer extension analysis for mapping the transcriptional start site of yqfS. Total RNA was isolated (34) from either vegetative (lane 1) or sporulating (stage T₇; lane 2) B. subtilis PERM317 cells grown in DSM. Primer extension was performed as described in Materials and Methods. The asterisk indicates the position of the primer extension product in the DNA sequence lying upstream of yqfS (see Fig. 1). The 5′ end of the yqfS transcript was determined by running a DNA sequencing ladder generated with the same primer (lanes G, A, T, and C) and was labeled with an arrowhead.

FIG. 8.

Comparison of the consensus Eσ^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 Eσ^G-type promoters (21, 43). The position of the mapped transcriptional start site of yqfS is indicated with an asterisk.

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 made in minimal Spizizen medium was divided into three subcultures; one (labeled “0”) was left untreated, and the other two were treated with either paraquat (PQ; 10 μM) or H₂O₂ (200 μM). The LB culture was treated in the same manner except that mitomycin C (MC; 0.5 μg/ml) was added to the culture. (C) B. subtilis YB3000 was grown in LB medium to an OD₆₀₀ of 0.5; at this point, the culture was equally divided, and mitomycin C (0.5 μg/ml) was added to one of the subcultures. In all cases, the β-galactosidase activity was determined with cell samples collected 2 h after the addition of the inducers.