Transition Phase Regulator AbrB Positively Regulates the sip1Ab1 Gene Expression in Bacillus thuringiensis

ABSTRACT Bacillus thuringiensis secreted insecticidal proteins (Sip) are a secretion that is toxic to coleopteran pests. However, the transcriptional mechanism of sip genes is still unknown. The transcriptional regulation of the sip1Ab1 gene and the expression of the Sip1Ab1 protein were investigated in this study. The results demonstrated that the secretion of the Sip1Ab1 protein in HD73 was almost the same as that in the original QZL38 strain during the transition phase. Analysis of the β-galactosidase activities of sip1Ab1-lacZ in both the HD73 and abrB mutant strains indicated that the transcription of sip1Ab1 is dependent on AbrB. Electrophoretic mobility shift assays showed that AbrB could bind with the sip1Ab1 promoter, and two binding sites of AbrB in the region of the promoter of sip1Ab1 were determined by DNase I footprinting assays. All of the above-described results proved that AbrB positively regulates the sip1Ab1 gene. IMPORTANCE Bacillus thuringiensis Sip proteins are secreted insecticidal toxins that are toxic to coleopteran pests. In this study, we investigated the transcriptional mechanism of the sip gene and showed strong evidence that Sip1Ab1 is secreted in the transition phase and that AbrB, a transition phase regulator that is usually a repressor, positively and directly regulates sip1Ab1. Reports of AbrB positive regulation are rare, even in Bacillus subtilis. To the best of our knowledge, no toxic gene has been reported to be positively regulated by AbrB in Bacillus species.

In Bacillus subtilis, AbrB is a global regulatory factor that regulates gene transcription in the log phase or transition phase (12). Purified AbrB protein binds specifically to fragments of DNA containing the promoters it affects (13). AbrB directly regulates more than 100 genes and influences hundreds more indirectly (14,15). AbrB mainly functions as a transcriptional repressor for gene transcription, but it also acts as an activator (16) involved in biofilm formation, antibiotic production, capacity development, extracellular enzyme production, motility, and sporulation (17)(18)(19)(20)(21)(22). Some examples of positive regulation of AbrB have been reported, such as scoC, rbs, and citB genes (16,17,23). Bacillus anthracis AbrB negatively regulates the toxin genes pagA, lef, and cya, which have higher transcriptional activity in the log phase (24), but the regulation is not direct (25).
The Sip1Ab1 proteins in strain QZL38 and their heterologous expression in HD73 were analyzed in this study. The transcription activities of the sip1Ab1 gene promoter in the HD73 wild-type strain and abrB mutant strain were analyzed using b-galactosidase. Electrophoretic mobility shift assays and DNase I footprinting assays showed the relationship between AbrB and the sip1Ab1 promoter.

RESULTS
Sip1Ab1 is secreted in the transition phase. In order to clarify the expression of the Sip1Ab1 protein in strain QZL38 and its heterologous expression in strain HD73, the promoter and open reading frame (ORF) of sip1Ab1 were ligated into the pHT304 vector and then electroporated into strain HD73. Time zero (T 0 ) was defined as the end of the exponential growth phase. The forespore septum was formed at 13 h after T 0 (T 13 ) (26). T 0 to T 13 covers the transition phase in LB medium of the HD73 strain. The same volume of the culture supernatants at T 0 , T 6 , T 12 , and T 18 in LB medium were collected and concentrated to 2 ml using a dialysis bag. The same loading volumes of strains QZL38 (Fig. 1A) and HD73 (Fig. 1B) bacteria in the culture supernatant were subjected to SDS-PAGE analysis. Through analysis of the protein characteristics and protein molecular weight, the protein bands indicated by the arrows in the figure were identified by mass spectrometry (see Materials and Methods) and were confirmed to be the Sip1Ab1 protein. The Sip1Ab1 protein had the highest expression at T 12 in strain QZL38, and was heterologously expressed in strain HD73. This result suggested that the Sip1Ab1 protein was regulated and expressed during the transition phase. This also means that the transition phase regulator may be involved in the expression of the sip1Ab1 gene. When we selected some global regulatory factors, namely, Spo0A, SigH, CcpA, and AbrB, and tested the regulatory relationship with sip1Ab1, only AbrB had a regulatory effect.
AbrB positively regulates the sip1Ab1 gene. Strain QZL38 contains 1 chromosome and 6 plasmids in, and the sip1Ab1 gene (RS27950) is located on plasmid 2. The main cry genes include cry8Ab-like, cry8Ca1, cry8Ea1, and cry8Fa1, and their positions on plasmid 2 are shown in Fig. 2A. To determine the transcriptional start site of the sip1Ab1 gene, a 59 rapid amplification of cDNA ends (RACE)-PCR experiment was performed (see Materials and Methods). The transcriptional start site (TSS) was confirmed to be a G located 36 nucleotides upstream of the QZL38 sip1Ab1 translational start codon (ATG) (Fig. 2B). The analysis of the QZL38 sip1Ab1 promoter region contains 235 (TAATATAA) and 210 (TTATAATTA) regions from the transcriptional start site (Fig. 2B).
To clarify the transcriptional mechanism of sip1Ab1, the HD DabrB strain was constructed using the principle of homologous recombination. A P sip1Ab1-L -lacZ fusion was constructed and transformed into the B. thuringiensis HD73 and HD DabrB strains. The amplified fragment (P sip1Ab1-L ) consisted of 567 bp upstream of the QZL38 sip1Ab1 ATG start codon ( Fig. 2A). The b-galactosidase activity assays (Fig. 2C) indicated that the transcriptional activities of P sip1Ab1-L in wild-type HD73 gradually increased between T 0 to T 10 . The transcriptional activity of P sip1Ab1-L was significantly abolished in the HD DabrB strain compared to that in the wild-type HD73 strain. This indicates that deletion of the abrB gene inhibits the transcriptional activity of the sip1Ab1 gene promoter. In other words, AbrB positively regulates the sip1Ab1 gene.
AbrB binds to the sip1Ab1 promoter. To test where AbrB directly regulates the sip1Ab gene, AbrB-His protein was purified via nickel column affinity chromatography. The desalting AbrB protein was dissolved in 20 mM Tris-HCl (pH 8.0).
A 567-bp promoter is too long for electrophoretic mobility shift assays (EMSA), so we first selected a region of the promoter (2201 to 136) (shown in Fig. 2A), P sip1Ab1-S , for EMSA. 6-carboxyfluorescein (FAM)-labeled fragments containing the promoter regions were incubated with increasing concentrations of AbrB. Protein-probe binding The two AbrB binding sites are indicated with underlines. (C) The activities of two sip1Ab1 promoters (P sip1Ab1-L and P sip1Ab1-S ) were assessed by lacZ fusions in HD73 and HD73 DabrB strains. Assays of b-galactosidase activity were performed to compare the activities of cells cultured in LB medium at 30°C with shaking at 220 rpm. T 0 is the end of the exponential growth phase, and T n means n hours after T 0 . Each value represents the mean of at least three independent replicates. Error bars show the standard deviations.
caused slow migration. Competitive gel shift assays were performed with labeled DNA probes and approximately 200-fold unlabeled DNA targets, respectively. As shown in Fig. 3A, 200-fold unlabeled DNA could dissociate most of the AbrB from the labeled promoter probe. The data shown in the figure confirm that AbrB directly binds to P sip1Ab1-S (Fig. 3A). To determine the AbrB binding site in the sip1Ab1 promoter, a DNase I footprinting assay was performed using the same promoter fragment used in the EMSA. A fragment (59-AGAATATTTCCTTTTATATACAATT-39) of P sip1Ab1-S was protected via AbrB binding (Fig. 3B), corresponding to the underlined sequence in the sip1Ab1 promoter region shown in Fig. 2B.
We compared the transcriptional activities of P sip1Ab1-S and P sip1Ab1-L . The results showed that the activity of P sip1Ab1-S was lower than that of P sip1Ab1-L in wild-type HD73, and it was also significantly abolished in the HD DabrB strain compared to that in the wild-type HD73 strain (Fig. 2C). We hypothesized that the promoter part P sip1Ab1-F (2531 to 2202) may contain an AbrB binding site. The ability of AbrB to bind to P sip1Ab1-F was examined via EMSA. The data shown in Fig. 3 confirmed that AbrB directly binds to P sip1Ab1-F (Fig. 3C), and the DNase I footprinting assay showed that a fragment (59-CATATGATGGAAGAGAAATACCAGTG-39) of P sip1Ab1-F was protected via AbrB binding (Fig. 3D), corresponding to the underlined sequence in the sip1Ab1 promoter region shown in Fig. 2B. This indicates that AbrB can bind with two regions of the sip1Ab1 promoter and directly regulate the sip1Ab1 gene.
To determine the roles of two AbrB binding sites, promoter fragments from 9531 to 136 from which binding site 1 or binding site 2 was deleted were fused to lacZ (Fig. 4A). The b-galactosidase activity assays (Fig. 4B) indicated that the transcriptional activity of P sip-delete1 in wild-type HD73 is no different from that of P sip1Ab1 -S. The transcriptional activity of P sip-delete2 was very low. The transcriptional activity of P sip-delete1 was significantly abolished in the HD DabrB strain compared to that in the wild-type HD73 strain.

DISCUSSION
Sip is a secreted protein, and Sip1Ab1 was secreted during the transition period ( Fig. 1). Sporulation medium lacking nutrients has also been used to produce the Sip1Ab1 protein, but production was extremely low (data not shown). The Sip1Ab1 protein was secreted in sufficient nutrient medium (Fig. 1). Thus, nutrient-poor medium is not suitable for Sip1Ab1 protein expression, and medium that can extend the transition phase is suitable for Sip1Ab1 expression.
In this study, AbrB positively regulated the toxin gene in B. thuringiensis (Fig. 2C). AbrB mostly negatively regulates gene transcription, and it is considered to be a repressor of gene expression, although several genes were positively regulated by AbrB in B. subtilis. All previous studies showed that AbrB negatively regulates gene expression in the B. cereus group. In B. cereus, overexpression of AbrB resulted in a nontoxin phenotype, and the Spo0A-AbrB circuit negatively regulated ces and repressed cereulide production (27). In B. anthracis, AbrB negatively regulates toxin genes (24). In B. thuringiensis, AbrB repressed biofilm formation and motility, which showed similarities to those in B. subtilis (28). AbrB also negatively regulated the immune inhibitor metalloprotease inhA1 (29). Currently, no toxin gene has been reported to be positively regulated by AbrB.
AbrB binds two regions of the sip1Ab1 promoter, and the binding sequences were confirmed here (Fig. 3). P sip1Ab1-F and P sip1Ab1-S each have a binding sites, and the difference in activity indicates that the two sites have distinct roles. A previous study reported that AbrB bound to the atxA promoter in B. anthracis, and the binding sequence was identified (25). Since both B. thuringiensis and B. anthracis belong to the B. cereus group, the homology of AbrB between them is 99% (see Fig. S1 in the supplemental material). Thus, we performed an alignment of the AbrB binding sequences of B. thuringiensis and B. anthracis. A conserved DNA sequence that was analyzed by MEME, TTGKWTAWAAARGGAA, was identified (see Fig. S2 in the supplemental material). The conserved sequence is not consistent with the AbrB consensus binding sequence, which in B. subtilis consists of bipartite TGGNA motifs separated by 4 to 5 bp (30), but they were all extremely AT rich. As Koehler mentioned, the B. anthracis and B. subtilis AbrB binding sites are also somewhat different (31). Via AbrB protein sequence alignment analysis, it was found that B. anthracis and B. thuringiensis AbrB proteins are 85% identical to B. subtilis AbrB (Fig. S1), and the last 32 residues are significantly different (24). AbrB probably recognizes a three-dimensional DNA structure rather than a typical DNA sequence (13,29,32,33). Here, we suggest that the AbrB consensus binding sequence in the B. cereus group is different from that in B. subtilis. Our results provide further understanding of the AbrB binding sequence and its regulatory mechanism.

MATERIALS AND METHODS
Strains, plasmids, and growth conditions. The strains and plasmids used in this study are summarized in Table 1. Escherichia coli strains DH5a and BL21 were used as hosts for molecular cloning and protein expression, respectively. E. coli SCS110 (also called ET12567) was used for transformation into B. thuringiensis cells, as described previously (34). B. thuringiensis HD73 was used as the recipient strain to monitor gene transcriptional activity and manipulate the gene cloning of B. thuringiensis (35,36). HD73 and its derivatives were routinely grown at 30°C in LB broth or on LB agar plates supplemented with either erythromycin (5 mg/ml) or kanamycin (100 mg/ml) when required. Secretion of Sip1Ab1 in QZL38 and HD73 strains. A single B. thuringiensis colony was inoculated in 5 ml LB medium and grown at 30°C with shaking overnight, and then 1 ml of the bacterial solution was added to 100 ml of LB medium and incubated to T 0 (T 0 is the end of the exponential growth phase, and T n is n hours after the end of the exponential growth phase), T 6 , T 12 , and T 18 . A 50-ml aliquot of bacterial solution was collected and centrifuged (4°C at 9,000 rpm for 10 min), and then the supernatants were collected into the dialysis bag in each period. An appropriate amount of polyethylene glycol (PEG) 8000 was also needed outside the dialysis bag. The concentration time was generally 6 to 10 h, and the temperature was 4°C. The proteins were dissolved in 2 ml of 20 mM Tris-HCl, and the same volume was analyzed via SDS-PAGE. The protein bands were cut from the gel and sent to a company (Beijing Protein Innovation) for mass spectrometry identification. After the protein gel was decolorized, it was hydrolyzed under the action of trypsin and detected by mass spectrometer (MicrOTOF-Q11; Bruker Daltonics).
Construction of the pHTsip1Ab1 and pHTP sip -lacZ expression vectors. The promoter sequences of the sip1Ab1 gene were amplified from QZL38 genomic DNA (GenBank accession no. CP032609; region, 192552 to 193646) using different primers (Table 2). A 567-bp fragment located from 2531 to 136 was PCR amplified from strain QZL38 with primers Psip1Ab1-L-F and Psip1Ab1-L-R, a 237-bp fragment located from 2201 to 136 was PCR amplified from strain QZL38 with primers Psip1Ab1-S-F and Psip1Ab1-L-R, a 329-bp fragment located from 2531 to 2202 was PCR amplified from strain QZL38 with primers Psip1Ab1-L-F and Psip1Ab1-F-R, and a 553-bp/554-bp fragment which contained 2531 to 136 except for binding site 1/binding site 2 was synthesized (Sangon Biotech, Shanghai) and ligated to the linearized pHT304-18Z plasmid, which contains a promoterless lacZ gene. A 1,598-bp fragment, containing the 567 bp upstream of the QZL38 sip1Ab1 ATG start codon and the sip1Ab1 open reading frame, was amplified and ligated to the linearized pHT304 plasmid. The recombinant plasmids were introduced into HD73. The resulting strains were placed on agar plates supplemented with erythromycin and verified by PCR.
b-Galactosidase activity analysis. The B. thuringiensis strains were cultivated in LB medium with shaking (220 rpm at 30°C). Aliquots (2 ml) of cultures were collected every 2-h interval from T 0 to T 10 (T 0 indicates the end of the exponential growth phase, and T n indicates n hours after T 0 ). The cells were centrifuged (13,000 Â g for 1 min), and the pellets were stored at 220°C until use. b-Galactosidase activities were measured as previously described (37) and are expressed as Miller units. The reported values represent the averages from at least three independent assays. 59 RACE analysis. The total RNA was extracted from the QZL38 cells grown in LB until the T 3 stage, and reverse transcription-PCR was conducted as previously described (38). We used the SMARTer RACE (switching mechanism at the 59 end of the RNA transcript-rapid amplification of cDNA ends) cDNA amplification kit (Clontech, Mountain View, CA) to determine the transcription start site, following the manufacturer's instructions. sip1Ab159race-R, located 200 bp downstream of the QZL38 sip1Ab1 start a FAM, 6-carboxyfluorescein. The restriction sites are underlined and bolded.
codon (ATG), was designed as the specific reverse primer. NestRace was the forward primer provided in the kit (Clontech, Mountain View, CA). sip1Ab159race-R and NestRace were used as specific primers for amplifying the 59 end of P sip1Ab1 cDNA. The sequences of the primers used in this study are shown in Table 2.
Screening of HD DabrB mutants. The HD73 abrB mutant was constructed using the principle of homologous recombination. The method is briefly described as follows. A 709-bp upstream region containing a 15-bp overlap with the 59 end of abrB (abrB fragment A) and a 729-bp downstream region containing a 21-bp overlap with the 39 end of abrB (abrB fragment B) were amplified from B. thuringiensis HD73 genomic DNA with the abrB-a/abrB-b and abrB-c/abrB-d primer sets, respectively. The kanamycin resistance gene (kan, 1,473 bp) was amplified with the Km-F/Km-R primer set using the DsigH mutant as a template. Subsequently, a long flanking PCR was performed with abrB fragment A, the kan fragment, and abrB fragment B as the templates, in that order, and the abrB-a/abrB-d primer set to generate a long fragment (2,828 bp). The resulting DNA fragment was doubly digested with BamHI and SalI and cloned into the erythromycin (ERY)-resistant, temperature-sensitive suicide plasmid pMAD. The recombinant plasmid was named pMADDabrB. The recombinant plasmid was transferred to E. coli ET for demethylation and then electroporated into strain HD73. The strain was subjected to high-temperature mutation at 37°C, and a strain named HD (pMADDabrB), with kanamycin resistance and no erythromycin resistance, was selected. Using the mutant cassette outer primers JDwabrB-1 and JDwabrB-2, the wild-type strain HD73 and the kanamycin-resistant and ERY-resistant strain were used as the templates to identify the mutant strain, and the obtained mutant strain was named HD DabrB.
Purification of the AbrB protein. The BL21 (pETabrB) strain was cultured in LB medium containing 100 mg/ml ampicillin at 37°C and 220 r/min to an optical density at 600 nm (OD 600 ) of 0.7 to 1.0 and then added to a final concentration of 0.5 mmol/liter isopropyl-b-D-thiogalactopyranoside (IPTG) induced at 18°C and 150 rpm for 12 h. The cells were collected by centrifugation at 9,000 rpm for 10 min at 4°C. The cells were suspended in 50 mM Tris-HCl buffer (pH 8.0). The suspension was ultrasonically disrupted on ice for 6 min (CP750, ultrasonic power 70%, ultrasound 3 s, and pause 5 s; Cole-Parmer). The supernatant and precipitate were separated by a low-temperature centrifuge (12,000 rpm for 10 min). The supernatant containing soluble AbrB protein was placed in a well-balanced nickel affinity chromatography column, and the His-tagged AbrB protein was fully combined with the column. Then, the protein was washed with 10 column volumes of equilibration buffer (20 mM/liter Tris-HCl [pH 8.0], 0.5 M/liter NaCl, and 20 mM/liter imidazole), and finally the proteins eluted by the 10 ml of elution buffer (20 mM/liter Tris-HCl [pH 8.0], 0.5 M/liter NaCl, and 250 mM/liter imidazole) were collected. SDS-PAGE was used to detect the eluted protein samples. The purified protein samples were desalted using the Äkta protein purifier, and the desalted protein was dissolved in 20 mM Tris-HCl (pH 8.0).
DNase I footprinting assay. The promoter region was PCR amplified with 2Â high-fidelity DNA polymerase premix (Tolo Biotech, Shanghai, China) from the plasmids pMAD19-TP sip1Ab1-F and pMAD19-TP sip1Ab1-S using M13F (FAM) and M13R primers to prepare the fluorescent FAM-labeled probes. The FAMlabeled probes were purified using the Wizard SV gel and PCR clean-up system (Promega, USA) and were quantified with a NanoDrop 2000C instrument (Thermo, USA). The DNase I footprinting assays were performed following Wang et al. (39).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, DOCX file, 0.2 MB.

ACKNOWLEDGMENTS
This work was supported by grants from the National Natural Science Foundation of China (grant 31530095) and the National Key Research and Development Program of China (grant 2017YFD0200400).
We declare no conflicts of interest.