The Regulation of Exosporium-Related Genes in Bacillus thuringiensis

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis (Bt) are spore-forming members of the Bacillus cereus group. Spores of B. cereus group species are encircled by exosporium, which is composed of an external hair-like nap and a paracrystalline basal layer. Despite the extensive studies on the structure of the exosporium-related proteins, little is known about the transcription and regulation of exosporium gene expression in the B. cereus group. Herein, we studied the regulation of several exosporium-related genes in Bt. A SigK consensus sequence is present upstream of genes encoding hair-like nap proteins (bclA and bclB), basal layer proteins (bxpA, bxpB, cotB, and exsY ), and inosine hydrolase (iunH). Mutation of sigK decreased the transcriptional activities of all these genes, indicating that the transcription of these genes is controlled by SigK. Furthermore, mutation of gerE decreased the transcriptional activities of bclB, bxpB, cotB, and iunH but increased the expression of bxpA, and GerE binds to the promoters of bclB, bxpB, cotB, bxpA, and iunH. These results suggest that GerE directly regulates the transcription of these genes, increasing the expression of bclB, bxpB, cotB, and iunH and decreasing that of bxpA. These findings provide insight into the exosporium assembly process at the transcriptional level.

Despite the extensive studies on the structure of the exosporium-related proteins, little is known about the transcription and regulation of exosporium gene expression in the B. cereus group. Herein, we demonstrate that the transcription of bclA, bclB, bxpA, bxpB, cotB, exsY, and iunH are controlled by RNA polymerase sigma factor SigK in Bt HD73. Furthermore, the expression of bclB, bxpA, bxpB, cotB, and iunH is directly regulated by GerE. gerE encodes the terminal transcription factor in the sporulation regulatory cascade in Bacillus subtilis. GerE is a small DNA-binding protein that is both an activator and a repressor in the mother cell that regulates the transcription of many genes involved in spore coat synthesis and assembly in the late stages of sporulation and germination [30][31][32] . GerE acts in conjunction with SigK-containing RNA polymerase to turn on the expression of the final class of sporulation genes. The appearance of GerE also switches off the expression of some genes that had been activated by SigK 31 .

Results
Transcriptional activity of hair-like nap protein genes. We identified 17 exosporium homologous genes with known functions in B. cereus and B. anthracis in Bt HD73 (Table 1) comprising genes encoding the hair-like nap proteins, basal layer proteins, and enzymes. A major component of the hair-like nap is the glycosylated collagen-like protein BclA. A second collagen-like protein, BclB, is also present in the exosporium 19 . In Bt HD73, HD73_1438 (bclA) and HD73_2664 (bclB) encode BclA and BclB and have 67.8% and 90.0% identity, respecively, to homologous genes in B. anthracis Sterne strain 7702 33 and B. cereus ATCC 10876 34 . To determine the transcription start site (TSS) of bclA and bclB, 5′ -RACE analysis was performed as described in the Methods. The TSSs of bclA and bclB were confirmed to be a single 5′ -end nucleotide residue C and G located 120 bp and 150 bp upstream of the start codon according to the sequences of 20 random clones, respectively (Figs 1A and 2A). Analysis of the bclA and bclB promoter sequences identified sequences CAC(-N 16 -)CATATGTTA and AGC(-N 16 -)CATATAATT upstream of the bclA and bclB TSS, respectively, which are similar to the consensus sequences recognized by SigK-containing RNA polymerase 35 , with the putative binding site centered at -10 and -35 nt with appropriate spacing (16 nt) between these consensus sequences (Figs 1A and 2A). SigK is a sigma factor that plays a role in the late stage of sporulation, and some SigK-dependent genes are negatively or positively regulated by GerE in the late stage of sporulation 31 . Thus, to study the transcription and regulation of the promoters PbclA and PbclB, PbclA-lacZ and PbclB-lacZ fusions were constructed and transformed into Bt wildtype strain HD73 and mutant strains, HD(Δ sigK) and HD(Δ gerE). The β -galactosidase assay showed that the transcriptional activity of PbclA was sharply decreased from T 10 to T 23 in HD(Δ sigK) (Fig. 1B). It was slightly increased from T 10 to T 18 in HD(Δ gerE), and with no significant difference from T 18 to T 23 compared with that of wild-type strain HD73 (Fig. 1B). However, the transcriptional activity of PbclB was sharply decreased from T 9 to T 23 both in HD(Δ sigK) and HD(Δ gerE) compared with that of HD73 (Fig. 2B). To determine whether GerE directly or indirectly regulates the PbclA and PbclB, GerE-GST protein was expressed in E. coli and purified. The ability of GerE to bind to a DNA fragment containing the PbclA (267 bp) and PbclB (276 bp) promoters was examined by EMSA. FAM-labeled fragments containing the promoter regions of bclB were incubated with different amounts of GerE and assayed for the formation of protein-DNA complexes. Slower-migrating probe-protein complexes were observed upon incubation with increasing amounts of GerE (Fig. 2C). It indicated that GerE recognizes and specifically binds to sequences within the bclB promoter fragment. To precisely determine the GerE-binding site in the bclB promoter, DNase I footprinting assays were carried out using the same bclB promoter fragment used in the EMSA (Fig. 2D). A 23-bp fragment corresponding to the boxed sequence in the bclB promoter region ( Fig. 2A) was protected by GerE binding. In sharp contrast, GerE did not bind to labeled bclA promoter (Additional file 1). This may result from the lack of direct binding, from a purified GerE protein partially defective in binding or from unfavorable in vitro binding conditions. These results indicated that transcription of PbclA and PbclB are controlled by SigK in the late stage of sporulation and that PbclB is directly activated by GerE, while PbclA is negatively regulated by GerE. Transcriptional activity of basal layer protein genes. We studied the transcription and regulation of four basal layer protein genes bxpA (HD73_2410), bxpB (HD73_1452), cotB (HD73_0469), and exsY (HD73_1449). These genes have 75.4%, 97.0%, 76.9%, and 87.0% identity, respectively, to homologous genes in B. anthracis or B. cereus ( Table 1). The TSSs of bxpA, bxpB, cotB, and exsY were confirmed to be a single 5′ -end nucleotide residue A, A, G, and G located 26 bp, 24 bp, 33 bp and 33 bp upstream of the start codon according to the sequences of 20 random clones, respectively (Figs 3A, 4A, 5A and 6A). Bioinformatics analysis predicted strong SigK-like consensus binding sequences upstream of the respective start codons of all four genes (Figs 3A, 4A, 5A and 6A). The β -galactosidase assay showed that the transcriptional activities of PbxpB and PcotB were abolished in HD(Δ sigK) and decreased in HD(Δ gerE) compared with those of wild-type strain HD73 (Figs 3B and 4B). The transcriptional activity of PbxpA was also abolished in HD(Δ sigK), whereas it was increased in HD(Δ gerE) compared with HD73 (Fig. 5B). EMSA showed that GerE could bind to the promoters of bxpB, cotB, and bxpA (Figs 3C, 4C and 5C). To precisely determine the GerE-binding site in the bxpB, cotB, and bxpA promoters, DNase I footprinting assays were carried out using the same promoter fragments used in the EMSA. A 37-bp, 23-bp and 31-bp fragments located on bxpB, cotB, and bxpA promoters were protected by GerE binding (Figs 3D, 4D and 5D) (corresponding to the boxed sequence in the bxpB, cotB, and bxpA regions shown in Figs 3A, 4A and 5A). The transcriptional activity of PexsY was sharply decreased in HD(Δ sigK) but showed no significant difference in HD(Δ gerE) (Fig.6B). These results indicated that transcription of PbxpA, PbxpB, PcotB, and PexsY is controlled by SigK in the late stage of sporulation and that PbxpA, PbxpB, and PcotB are directly regulated by GerE.
Transcriptional activity of the inosine hydrolase gene. Inosine hydrolase is encoded by iunH (HD73_3089) in Bt HD73, which has 93.1% identity to the homologous gene bas2693 in the B. anthracis Ames strain 15 . According to the sequences of 20 random clones, the TSSs of iunH was confirmed to be a single 5′ -end nucleotide residue G residue located 10 bp upstream of the start codon (Fig. 7A). SigK consensus binding site was present upstream of iunH (Fig. 7A). The β -galactosidase assay showed that the transcriptional activity of PiunH was abolished from T 8 to T 22 in HD(Δ sigK) and lower in HD(Δ gerE) than in HD73 (Fig. 7B). EMSA showed that GerE could bind to the iunH promoter (Fig. 7C) and DNase I footprinting assays showed that a 15-bp fragment was protected by GerE binding (Fig. 7D) (corresponding to the boxed sequence in the iunH region shown in Fig. 7A), together suggesting that transcription of iunH is controlled by SigK and is directly regulated by GerE.

Discussion
In a B. subtilis mother cell, a regulatory network with a cascade of four transcription factors (SigE, SpoIIID, SigK, and GerE) controls gene expression in the mother cell during sporulation 36 . SigE and SigK are sigma subunits of RNA polymerase. SpoIIID and GerE, two small DNA-binding proteins, repress or activate transcription of many mother cell genes 31,37 . SigK directs the expression of most genes encoding coat structural components and factors required for spore germination, and mother-cell lysis 38 . The decisive role of SigK in spore coat assembly is evidenced by the large number of genes encoding coat structural components found in the SigK regulon 4,38 . Unlike the coat that constitutes the outermost layer of the mature B. subtilis spore 6 , the B. cereus group species are encircled by the exosporium 5 . Little is known about the transcription and regulation of the expression of exosporium genes in the B. cereus group. Indeed, only exsB is known to undergo SigK-mediated transcription and is positively regulated by GerE, as shown in our pervious study 39 . In this study, we first confirmed that the transcription of exosporium-related genes bclA, bclB, bxpA, bxpB, cotB, exsY, and iunH are controlled by SigK using a β -galactosidase assay. The SigK consensus sequence is located upstream of these and ten other exosporium-related genes in Bt and is predicted to be present in most B. cereus group strains (Additional file 2). This finding suggested that the transcription mechanisms of exosporium genes are similar throughout the B. cereus group.
In the B. subtilis cascade, the synthesis of each factor depends upon the activity of the prior factor, and there is a feedback loop in which SigK RNAP transcribes gerE, which then negatively regulates transcription of the sigK gene 31,40 . Some SigK-dependent genes such as oxalate decarboxylase encoded gene oxdD 41 and the germination gene gerT 30 are negatively regulated by GerE. In contrast, other SigK-dependent genes encoding spore coat proteins such as cotB 31 , cotC 31 , yxeE 42 , and yeeK 43 are positively regulated by GerE in B. subtilis. We observed similar effects under the current conditions. The transcription of bclA and bxpA is negatively regulated by GerE, which could bind to the promoter of bxpA. Furthermore, the transcription of bclB, bxpB, cotB, and iunH is positively regulated by GerE, and their promoters could bind to GerE.
The collagen-like glycoproteins BclA and BclB require BxpB to assemble the hair-like nap of exosporium, and the assembly timing of the three proteins is similar 19 . Based on transcriptional level, we demonstrated that transcription of these three genes occurs nearly at the same stage (T 10 ). BxpA is located below the spore coat associated with the cortex and is synthesized during sporulation and assembled into the spore before mother cell lysis, but it is not found in vegetative cells in B. anthracis Ames 44 . Furthermore, the SigK consensus sequence is found upstream of bxpA 13 . We provide new evidence that transcription of bxpA initiates at T 8 and is abolished in the sigK mutant. ExsY is a homologue of B. subtilis cysteine-rich spore coat proteins CotY and CotZ 45 , that participates in assembly of an intact exosporium 21 . The time of synthesis of ExsY protein in the sporulation phase was detected by western-blot 21 . We confirmed that transcription of exsY begins at T 7 under the control of SigK and is similar to the transcriptional mechanism of cotYZ in B. subtilis 46 . CotB is similar to ExsY in B. anthracis 47 and has 30% amino acid identity to B. subtilis spore coat protein CotB 48 . We confirmed that the transcription of cotB begins at T 10 under the control of SigK, and is regulated by GerE in Bt. The manner of transcription and regulation is similar between Bt and B. subtilis 31,36 . The transcriptional pattern of bclA, bxpB, cotB, bxpA, exsY, and iunH in wild-type HD73 is very similar, increasing from T 8 to T 17 and decreasing thereafter, suggesting that these proteins are assembled into the basal layer and hair-like nap simultaneously and are nearly complete at T 17 . However, the transcription of bclB is significantly higher than that of blcA after T 17 with continuous transcriptional activity from T 8 to T 23 . These transcriptional data are differ to previous reports, which have suggested that bclB and bclA are transcribed at an identical stage in sporulation, but with bclB transcribed at an approximately two-fold lower level 9,49 . The present data provide evidence that transcription of some exosporium genes is controlled by SigK and partially regulated by GerE. These findings provide insight into the exosporium assembly process at the transcriptional level.  Table 2. Bt strain HD73 was used throughout the study (accession numbers CP004069) 50 . Escherichia coli strain TG1 was used as the host for cloning experiments. The Dam -/Dcm -E. coli ET12567 strain (laboratory stock) was used to generate unmethylated DNA for the electrotransformation assay. Bt strains were transformed by electroporation, as described previously 51,52 . E. coli and Bt strains were cultured in Luria-Bertani (LB) medium, with 220 rpm shaking at 37 °C and 30 °C, respectively. The antibiotic concentrations used for bacterial selection were as follows: 100 μ g/ml kanamycin and 10 μ g/ml erythromycin for Bt, and 100 μ g/ml ampicillin for E. coli.

DNA manipulation techniques. PCR was performed using Taq and KOD DNA polymerase (New England
BioLabs Ltd., Beijing, China). Amplified fragments were purified using purification kits (Axygen, Union City, CA, USA). Bt chromosomal DNA was extracted with the Puregene kit (Gentra, Minneapolis, MN, USA). Restriction enzymes and T4 DNA ligase (TaKaRa Biotechnology, Dalian, China) were used according to the manufacturer's instructions. Oligonucleotide primers (Table 3) were synthesized by Sangon (Shanghai, China). E. coli plasmid DNA was extracted using the Axygen Plasmid Extraction Kit. All constructs were confirmed by DNA sequencing (BGI, Beijing, China).
Total RNA isolation and 5′-RACE analysis. For total RNA purification, strain HD73 was grown as previously described in SSM medium until the T14 stage of stationary phase (corresponding to 14 h after the end of the exponential phase) 53 . cDNA synthesis and transcriptional start sites (TSSs) of the exosporium genes were determined using the SMARTer TM RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA) according to the manufacturer's instructions. Gene-specific primers and the universal primer mix (UPM) ( Table 3) were used to amplify the 5′ end of exosporium genes mRNA.
Expression and purification of GerE. GerE protein with a glutathione S-transferase (GST) tag was purified from E. coli 54 . The E. coli BL21(DE3) strain carrying pGEXgerE plasmid was incubated in LB medium. When   the optical density at 600 nm (OD600) reached 0.6, IPTG was added to a final concentration of 1 mM. After 4 h of induction at 37 °C, the bacterial cells were harvested by centrifuging the culture at 13,000 × g for 10 min. The pellet was resuspended in phosphate-buffered saline (PBS) and sonicated on ice. All subsequent procedures were carried out at 4 °C. The supernatant was collected by centrifuging the lysate at 13,000 × g for 20 min and loading it onto a glutathione-Sepharose 4B column previously equilibrated with PBS buffer. The column was washed with 50 mM Tris-HCl containing 10 mM reduced glutathione (pH 8.0). The fractions were analyzed by SDS-PAGE. Fractions with the target protein were pooled and dialyzed against PBS buffer. The purified GST-GerE protein was analyzed by SDS-PAGE on a 12% polyacrylamide gel with a protein molecular standard. All the steps described above were performed according to the manufacturer's instructions (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK).
Gel mobility shift assays. The DNA fragment was obtained by PCR of strain HD73 genomic DNA using specific primers (Table 3) labeled with a fluorescent 5′ -end 6-FAM modification and confirmed by DNA sequencing. Electrophoresis mobility shift assays (EMSA) were performed as previously described 55 to analyze the binding of purified GerE protein to the promoter of exosporium genes. Briefly, the DNA probe (0. Tris-base, 90 mM boric acid, 2 mM EDTA, pH 8.0) for resolution of the complexes using a Mini-PROTEAN system (Bio-Rad) at 160 V for 1 h. Signals were visualized directly from the gel with the FLA Imager FLA-5100 (Fujifilm). The specificity of the shift was confirmed using poly(dI:dC), GST protein, and bovine serum albumin (BSA); the cry1Ac promoter (which does not bind to GerE protein; data not shown) was used as the negative control.
DNase I footprinting assays. DNase I footprinting assays were performed based on a fluorescence labeling procedure 56 . Briefly, the promoters DNA of exosporium genes were PCR-amplified using the fluorescently labeled primers and purified from an agarose gel. The labeled DNA probe (400 ng) was incubated for 30 min at 25 °C with the different amounts of GerE in a total volume of 40 μ l binding buffer (described above for EMSA). DNase I digestion was then performed for 1 min at 25 °C and stopped with stop buffer (Promega). After phenol-chloroform extraction and ethanol precipitation, the samples were loaded on an Applied Biosystems 3730 DNA genetic analyzer with an internal-lane size standard (ROX-500, Applied Biosystems). A dye primer-based sequencing kit (Thermo) was used to precisely determine the sequences after their alignment wtih capillary electrophoresis results. Electropherograms were analyzed with GeneMarker v1.8 (Applied Biosystems).
Construction of the promoters of exosporium genes with lacZ gene fusion. The promoters of exosporium genes were amplified from Bt HD73 genomic DNA using specific primers. Promoter restriction fragments were then ligated into the pHT304-18Z vector containing a promoterless lacZ gene 57 . Recombinant pHT-Pn (where n indicates the name of exosporium genes) was introduced into Bt HD73, ΔsigK and ΔgerE mutant strains. The resultant strains, HD73(Pn), ΔsigK(Pn), and ΔgerE(Pn), were selected by resistance to erythromycin and tested by PCR to confirm the presence of the promoter fragments in the plasmids.
β-Galactosidase assays. Bt strains containing lacZ transcriptional fusions were cultured in Schaeffer's sporulation medium (SSM) 58 at 30 °C and 220 rpm. A 2-ml volume was collected at 1-h intervals from T 8 to T 22 (T 0 is the end of the exponential phase, and T n is n hours after T 0 ), from which cells were harvested by centrifugation for 1 min at 10,000 × g. The supernatant was removed, and the pellet was stored at − 20 °C or resuspended in 500 μ l Buffer Z (0.06 M Na 2 HPO 4 , 0.04 M NaH 2 PO 4 , 0.01 M KCl, 1 mM MgSO 4 ) with 1 mM dithiothreitol. The β -galactosidase activity was determined as previously described 59 and expressed as Miller units. Reported values represent averages from at least three independent assays.