A Novel Regulator PepR Regulates the Expression of Dipeptidase Gene pepV in Bacillus thuringiensis

Bacillus thuringiensis produces insecticidal crystal proteins encoded by cry or cyt genes and targets a variety of insect pests. We previously found that a strong promoter of a DeoR family transcriptional regulator (HD73_5014) can efficiently drive cry1Ac expression in B. thuringiensis HD73. Here, we investigated the regulation of neighbor genes by HD73_5014. The HD73_5014 homologs are widely distributed in Gram-positive bacterial species. Its neighbor genes include pepV, rsuA, and ytgP, which encode dipeptidase, rRNA pseudouridine synthase and polysaccharide biosynthesis protein, respectively. The four open reading frames (ORFs) are organized to be a pepR gene cluster in HD73. RT-PCR analysis revealed that the rsuA and ytgP genes formed a transcriptional unit (rsuA-ytgP operon), while pepV formed a transcriptional unit in HD73. Promoter-lacZ fusion assays showed that the pepV and rsuA-ytgP promoters are regulated by HD73_5014. EMSA experiments showed that HD73_5014 directly binds to the pepV promoter region but not to the rusA-ytgP promoter region. Thus, the HD73_5014 transcriptional regulator, which controls the expression of the dipeptidase pepV, was named PepR (dipeptidase regulator). We also confirmed the direct regulation between PepR and PepV by the increased sensitivity to vancomycin in ΔpepV and ΔpepR mutants compared to HD73.


Introduction
Bacillus thuringiensis (Bt) is a very important microorganism in the biological control of plant pests and diseases [1].During sporulation, it can produce crystals which are mainly formed by the insecticidal crystal proteins and are toxic to many pests, including more than 500 species in nine orders such as Lepidoptera, Hymenoptera, Diptera, Coleoptera, Trichoptera, Orthoptera, and so on [2].As of an update dated 22 February 2024, approximately 858 cry and cyt genes encoding crystal proteins have been discovered (http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/).The cry genes express during the stationary phase, and crystal production constitutes 20 to 30% of the dry weight of sporulating cells [3].
Several cry and non-cry gene promoters have been identified to effectively direct cry gene expression.For example, the cry1Ac promoter can direct the expression of a variety of cry-like genes [4], such as cry1Ac, cry1Ab, cry1Ac5, cry1Ac-av3, cry2Ab27, and cry8.Similarly, the expression of Cry1AbMod/Cry1AcMod [5], Cry1Ac [6], Cry1c [7], Cry3A [8], Cry8Ga [9], and Cry69Aa1 [10] can be directed by the cry3A promoter.Recent reports have shown that some non-cry gene promoters with high-level activity were utilized for the expression of cry genes [11,12].PexsY is a strong promoter of the exosporium basal layer structural gene exsY in late sporulation which has been used to express cry1Ac genes in Bt [11].P5014, a non-cry gene strong promoter controlled by Sigma E, was used to strongly direct cry1Ac expression in Bt HD73.The HD73_5014 gene with promoter P5014 was annotated as the DeoR family transcriptional regulator [12].However, the function and the target gene of HD73_5014 as transcriptional regulator are still unknown.
In the HD73 genome, the upstream of the HD73_5014 gene was the pepV gene, which encodes a dipeptidase.Dipeptidases are involved in the final breakdown of protein degradation fragments produced by other peptidases (e.g., aminopeptidasesare PepN, PepC, PepP, PepX, PepA [13] and endopeptidasesare PepO1, PepO2, PepF1 and PepF2 [14,15]).In Lactococcus lactis MG1363, PepV has been found to be involved in resistance to vancomycin.The transcription of pepV in L. helveticus was regulated by a CodY-like regulator and the BCAA-responsive transcriptional regulator which binds adjacent to the pepV promoter region [16,17].However, whether PepV had a similar function and its transcription regulated by PepR (HD73_5014, encoding dipeptidase regulator) required further investigation in B. thuringiensis.

DNA Manipulation and Transformation
Plasmid DNA was extracted from E. coli cells with a Plasmid Miniprep Kit (Axygen, Beijing, China).Restriction enzymes and T4 DNA ligase (Takara Biotechnology Corporation, Dalian, China) were used according to the manufacturer's instructions.PCR was performed with high-fidelity PrimeSTAR HS DNA polymerase (Takara Biotechnology Corporation, Dalian, China) or Taq DNA polymerase (BioMed, Beijing, China).DNA fragments were purified from 1% agarose gels using an AxyPrep DNA Gel Extraction Kit (Axygen).Standard procedures were followed for E. coli transformation [25], and Bt HD73 cells were transformed by electroporation, as previously described [26].

RNA Isolation and Reverse Transcription PCR (RT-PCR) of pepR Neighbor Genes
The HD73 strain was cultured in SSM at 30 • C and harvested at T 6 .Total RNA was extracted using the RNAprep Pure Bacteria Kit (Aidlab, Beijing, China).The RNA (500 ng) was used for reverse transcription using the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China).The primers were used to detect the expression of pepR and neighbor genes locus, as shown in Table 2. To confirm the absence of DNA contamination, all RNA samples were routinely subjected to 16S rRNA gene PCR using 16S rRNA-F/16S rRNA-R primers.

Primer Name
Sequence (5 a Restriction enzyme sites are underlined.
To detect the transcriptional activity of the PpepV and PrsuA-ytgP promoters in HD73 and ∆pepR strains, HD (PpepV), ∆pepR (PpepV), HD (PrsuA-ytgP), and ∆pepR (PrsuA-ytgP) were cultured in SSM medium at 30 • C with shaking 220 rpm.Two milliliters of culture were collected at 1 h intervals from T 0 to T 7 (T 0 indicates the end of the exponential growth phase and T n indicates n hours after time T 0 ).The cells were centrifuged (12,000× g, 1 min), and the pellets were stored at −20 • C until use.β-galactosidase activities were measured as previously described [24].Values are reported as the mean and standard error of at least three independent assays.

Expression and Purification of PepR
The pETpepR plasmid containing pepR from HD73 was constructed by amplifying pepR with primers pETpepR-F/pETpepR-R and cloning into BamHI/SalI-digested pET21b.The pETpepR was transferred into the E. coli BL21(DE3), and the positive transformants were BL21 (pETpepR).The transformants were grown to the OD 600nm of approximately 1.0 in LB medium supplemented with ampicillin; then, they were incubated with isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 1 mM in an 18 • C shaking incubator for 12 h.Total cell proteins from each optimization experiment were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The expression and purification of the PepR-His protein was performed as previously described [27].

Electrophoresis Mobility Shift Assays
The pepV fragment was PCR amplified from HD73 genomic DNA by specific primers labeled with a 5 ′ -end FAM modification and confirmed by pepV sequencing.Electrophoresis mobility shift assays (EMSA) [28] were performed as described to analyze the binding of purified PepR.

Vancomycin Sensitivity Assay
The ∆pepR, ∆pepV, and HD73 strains were grown in LB medium until they reached an optical density of approximately 1.0 at 600 nm (OD 600 nm).Then, each strain suspension was divided into triplicates and transferred to 100 mL of SSM medium with a vancomycin concentration of 6 ng/mL.The OD 600 nm was measured every hour at 30 • C.

PepR Homologs Are Widely Distributed in Various Gram-Positive Bacteria
In a previous study, we found that the pepR gene promoter can efficiently direct cry1Ac expression in HD73 [12].However, the function of the pepR gene and the target gene of PepR as a transcriptional regulator are still unknown.In this study, we analyzed PepR homologs through a phylogenetic evolutionary tree.PepR homologs with amino acid similarity greater than 70% and 100% amino acid coverage from the NCBI database were screened (Table S1).The PepR homologs were searched with an E-value lower than 2.07 × 10 −45 .The phylogenetic tree showed that the PepR homologs were present in 143 bacterial species, all of which were Gram-positive and mainly included Anoxybacillus spp., Bacillus spp., Caldibacillus spp., Cytobacillus spp., Domibacillus spp., Ectobacillus spp., Heyndrickxia spp., Jeotgalibacillus spp., Kurthia spp., Lysinibacillus spp., Metabacillus spp., Neobacillus spp., Paenisporosarcina spp., Peribacillus spp., Planococcus spp., Psychrobacillus spp., Sporosarcina spp., and Sutcliffiella spp., etc. (Figure 1).This finding suggests that PepR homologs are highly conserved across a wide range of Gram-positive bacteria.

Characterization of the Transcription Units in the pepR Gene Cluster
The nucleotide sequence of the pepR gene cluster (4489 bp) of HD73 is comprised of four ORFs, which are annotated as dipeptidase (pepV, HD73_5013), DeoR family transcriptional regulator (pepR, HD73_5014), rRNA pseudouridine synthase (rsuA, HD73_5015), and polysaccharide biosynthesis protein (ytgP, HD73_5016) (Figure 2A).To determine the transcription units of the pepR cluster, a series of primers were designed.The total RNA was extracted at T6 from B. thuringiensis HD73 cultures grown in SSM.RT-PCR results showed mRNA overlapping the rsuA and ytgP genes (Figure 2B).However, no mRNA overlapping thpR and pepV was transcribed.These results indicate that rsuA and ytgP genes form a transcriptional unit (rsuA-ytgP operon), while thpR and pepV each form a transcriptional unit independently.

Characterization of the Transcription Units in the pepR Gene Cluster
The nucleotide sequence of the pepR gene cluster (4489 bp) of HD73 is comprised of four ORFs, which are annotated as dipeptidase (pepV, HD73_5013), DeoR family transcriptional regulator (pepR, HD73_5014), rRNA pseudouridine synthase (rsuA, HD73_5015), and polysaccharide biosynthesis protein (ytgP, HD73_5016) (Figure 2A).To determine the transcription units of the pepR cluster, a series of primers were designed.The total RNA was extracted at T 6 from B. thuringiensis HD73 cultures grown in SSM.RT-PCR results showed mRNA overlapping the rsuA and ytgP genes (Figure 2B).However, no mRNA overlapping thpR and pepV was transcribed.These results indicate that rsuA and ytgP genes form a transcriptional unit (rsuA-ytgP operon), while thpR and pepV each form a transcriptional unit independently.

PepR Positively Regulates pepV and rsuA-ytgP
To verify whether PepR regulates the transcription of pepV and rsuA-ytgP, we first constructed a pepR deletion mutant.The upstream (pepR-A, 1065 bp) and downstream (pepR-B, 1057 bp) homologous fragments of pepR were amplified from the HD73 genome DNA.Then, the pepR-A, pepR-B, and kanamycin resistance gene (pepR-K, 1506 bp) fragments were ligated together by overlapping PCR.The resulting 3563-bp fragment was inserted into the temperature-sensitive pMAD plasmid, leading to pMADΩDpepR plasmid.The pMADΩDpepR plasmid was electroporated into HD73.This transformant was then screened at 37 °C to identify the pepR deletion mutant, which lacked erythromycin resistance but was resistant to kanamycin.The diagram shows that the kanamycin resistance gene on the recombinant plasmid was exchanged by homolog recombination with pepR on the HD73 chromosome (Figure 3A).HD73 and ΔpepR strains were confirmed by PCR using primers of pepR-WF/pepR-WR.PCR products with the size of 3677 bp from ΔpepR and 2372 bp from HD73 were detected by agarose gel electrophoresis (Figure 3B).The ΔpepR DNA as template was amplified by PCR using primers (pMAD-F/pMAD-R) for detecting the pMAD plasmid, resulting in no PCR products (Figure 3B).It proved that the ΔpepR mutant was obtained.

PepR Positively Regulates pepV and rsuA-ytgP
To verify whether PepR regulates the transcription of pepV and rsuA-ytgP, we first constructed a pepR deletion mutant.The upstream (pepR-A, 1065 bp) and downstream (pepR-B, 1057 bp) homologous fragments of pepR were amplified from the HD73 genome DNA.Then, the pepR-A, pepR-B, and kanamycin resistance gene (pepR-K, 1506 bp) fragments were ligated together by overlapping PCR.The resulting 3563-bp fragment was inserted into the temperature-sensitive pMAD plasmid, leading to pMADΩDpepR plasmid.The pMADΩDpepR plasmid was electroporated into HD73.This transformant was then screened at 37 • C to identify the pepR deletion mutant, which lacked erythromycin resistance but was resistant to kanamycin.The diagram shows that the kanamycin resistance gene on the recombinant plasmid was exchanged by homolog recombination with pepR on the HD73 chromosome (Figure 3A).HD73 and ∆pepR strains were confirmed by PCR using primers of pepR-WF/pepR-WR.PCR products with the size of 3677 bp from ∆pepR and 2372 bp from HD73 were detected by agarose gel electrophoresis (Figure 3B).The ∆pepR DNA as template was amplified by PCR using primers (pMAD-F/pMAD-R) for detecting the pMAD plasmid, resulting in no PCR products (Figure 3B).It proved that the ∆pepR mutant was obtained.
Microorganisms 2024, 12, x FOR PEER REVIEW 8 of 13 the transcriptional activity of PpepV was significantly reduced from T0 to T7 in ∆pepR (Figure 3C).The result revealed that the PpepV promoter is regulated by PepR.Similarly, the transcriptional activity of PrsuA-ytgP in the pepR mutant strain was also significantly decreased compared to that in HD73 from T0 to T7 (Figure 3D), which suggested that the rsuA-ytgP operon was also regulated by PepR.

PepR Binds to the pepV Promoter
To determine whether the PepR protein directly binds to the pepV or rsuA-ytgP promoter regions, the electrophoretic mobility shift assay (EMSA) was performed.We Then, PpepV and PrsuA-ytgP promoter activity was measured in HD73 and ∆pepR.The PpepV and PrsuA-ytgP promoters were fused with the lacZ reporter gene and transformed into HD73 and ∆pepR, respectively.β-galactosidase activity showed that the activity of PpepV increased from T 0 to T 7 and reached the highest level at T 7 in HD73.However, the transcriptional activity of PpepV was significantly reduced from T 0 to T 7 in ∆pepR (Figure 3C).The result revealed that the PpepV promoter is regulated by PepR.Similarly, the transcriptional activity of PrsuA-ytgP in the pepR mutant strain was also significantly decreased compared to that in HD73 from T 0 to T 7 (Figure 3D), which suggested that the rsuA-ytgP operon was also regulated by PepR.

PepR Binds to the pepV Promoter
To determine whether the PepR protein directly binds to the pepV or rsuA-ytgP promoter regions, the electrophoretic mobility shift assay (EMSA) was performed.We constructed the recombinant plasmid, pETpepR, which was able to express the His-tagged PepR protein and introduced it into the E. coli BL21(DE3).The PepR-His protein with a molecular weight of approximately 10.06 kDa was expressed in E. coli BL21(pETpepR) and purified by Ni 2+ -affinity chromatography (Figure 4A).Then, we tested the binding of the PepR protein to the pepV or rsuA-ytgP promoter regions.A 0.21 nM PpepV-labeled probe was used to bind to PepR (low concentrations of 34.79 µM).The PpepV-labeled probe at 0.21 nM was strongly shifted with a 44.73 nM PepR (Figure 4B).Notably, a 200-fold excess of unlabeled probe competed with the labeled probe, confirming the specific binding (Figure 4B).These results demonstrated that PepR directly binds to the pepV promoter region.However, when 0.19 nM of the PrsuA-ytfP probe was exposed to PepR concentrations ranging from 14.91 µM to 44.73 µM, no bind-shift could be detected, indicating that PepR cannot interact with the rusA-ytfP promoter region (Figure 4C).These results strongly support the theory that PepR directly regulates the expression of pepV and indirectly regulates rusA-ytfP expression.
Microorganisms 2024, 12, x FOR PEER REVIEW 9 of 13 constructed the recombinant plasmid, pETpepR, which was able to express the His-tagged PepR protein and introduced it into the E. coli BL21(DE3).The PepR-His protein with a molecular weight of approximately 10.06 kDa was expressed in E. coli BL21(pETpepR) and purified by Ni 2+ -affinity chromatography (Figure 4A).Then, we tested the binding of the PepR protein to the pepV or rsuA-ytgP promoter regions.A 0.21 nM PpepV-labeled probe was used to bind to PepR (low concentrations of 34.79 µM).The PpepV-labeled probe at 0.21 nM was strongly shifted with a 44.73 nM PepR (Figure 4B).Notably, a 200-fold excess of unlabeled probe competed with the labeled probe, confirming the specific binding (Figure 4B).These results demonstrated that PepR directly binds to the pepV promoter region.However, when 0.19 nM of the PrsuA-ytfP probe was exposed to PepR concentrations ranging from 14.91 µM to 44.73 µM, no bind-shift could be detected, indicating that PepR cannot interact with the rusA-ytfP promoter region (Figure 4C).These results strongly support the theory that PepR directly regulates the expression of pepV and indirectly regulates rusA-ytfP expression.

ΔpepR and ΔpepV Mutants Are More Sensitive to Vancomycin than HD73
To characterize the function of the pepV gene, we constructed a pepV deletion mutant.The homolog arms on both sides of pepV were pepV-A (993 bp) and pepV-B (1023 bp) amplified from HD73 using primers pepV-AF/pepV-AR and pepV-BF/pepV-BR, respectively.The pepV-K fragment was obtained using PCR with pepV-KF/pepV-KR.The overlapping PCR products of pepV-A, pepV-B, and pepV-K were amplified with pepV-AF/pepV-BR primers, resulting in a 3452 bp fragment.The fragment was digested with BamHI and EcoRI sites and ligated to the temperature-sensitive vector of pMAD to obtain recombinant plasmid pMAD∆pepV.The recombinant plasmid pMAD∆pepV was electroporated into HD73.Transformants were selected for anti-sensitivity to erythromycin and kanamycin (Figure 5A).Positive transformants were verified at 37 °C.Colonies lacking erythromycin resistance and containing kanamycin resistance were selected for ∆pepV.
The pepV deletion mutant and HD73 strains were confirmed by PCR using pepV-  To characterize the function of the pepV gene, we constructed a pepV deletion mutant.The homolog arms on both sides of pepV were pepV-A (993 bp) and pepV-B (1023 bp) amplified from HD73 using primers pepV-AF/pepV-AR and pepV-BF/pepV-BR, respectively.The pepV-K fragment was obtained using PCR with pepV-KF/pepV-KR.The overlapping PCR products of pepV-A, pepV-B, and pepV-K were amplified with pepV-AF/pepV-BR primers, resulting in a 3452 bp fragment.The fragment was digested with BamHI and EcoRI sites and ligated to the temperature-sensitive vector of pMAD to obtain recombinant plasmid pMAD∆pepV.The recombinant plasmid pMAD∆pepV was electroporated into HD73.Transformants were selected for anti-sensitivity to erythromycin and kanamycin (Figure 5A).Positive transformants were verified at 37 • C. Colonies lacking erythromycin resistance and containing kanamycin resistance were selected for ∆pepV.
It has been demonstrated that PepV is related to vancomycin resistance in L. lactis MG1363 [29].A vancomycin resistance test was performed by inoculating ∆pepR, ∆pepV, and HD73 in SSM medium with 6 ng/mL concentrations of vancomycin.The result showed that the absence of vancomycin had no effect on the growth of ∆pepR, ∆pepV, and HD73 strains.However, the addition of vancomycin slowed HD73 growth.∆pepV growth stagnated while ∆pepR lysed and died after vancomycin was added (Figure 6).The result suggests that ∆pepR and ∆pepV were more sensitive to vancomycin than HD73, and ∆pepR was more sensitive to vancomycin than ∆pepV.The pepV deletion mutant and HD73 strains were confirmed by PCR using pepV-WF/pepV-WR primers.PCR was performed with HD73 and ∆pepV chromosomal DNA as template.The product from ∆pepV was a 3794 bp fragment, while the product from HD73 was a 3692 bp fragment (Figure 5B, Lanes b and c).The ∆pepV mutant was confirmed using PCR with pMAD plasmid primers of pMAD-F/pMAD-R, which did not produce any PCR product (Figure 5B, Lane d).In addition, PCR products were generated using HD73 and ∆pepV chromosomal DNA as templates with primers (pepV-WF/pepV-KR).There was a 2713 bp fragment from ∆pepV, while no product from HD73 was obtained (Figure 5B, Lanes f and e).The result confirms that the ∆pepV mutant was successfully obtained.
It has been demonstrated that PepV is related to vancomycin resistance in L. lactis MG1363 [29].A vancomycin resistance test was performed by inoculating ∆pepR, ∆pepV, and HD73 in SSM medium with 6 ng/mL concentrations of vancomycin.The result showed that the absence of vancomycin had no effect on the growth of ∆pepR, ∆pepV, and HD73 strains.However, the addition of vancomycin slowed HD73 growth.∆pepV growth stagnated while ∆pepR lysed and died after vancomycin was added (Figure 6).The result suggests that ∆pepR and ∆pepV were more sensitive to vancomycin than HD73, and ∆pepR was more sensitive to vancomycin than ∆pepV.
frame deletion pepV mutant.The pepV gene was substituted by a kanamycin-resistant gene through double homolog recombination events.(B) Identification of the ΔpepR knockout mutant by PCR.PCR products were amplified from HD73 and ΔpepV strains using the primer pairs of pepV-WF/pepV-WR (Lanes b and c) and pepV-WF/pepV-KR (Lanes e and f).To confirm the presence of the pMADΩDpepV plasmid in the ΔpepV genome, PCR was performed using pMAD plasmid universal primers (pMAD-F/pMAD-R) (Lane d).The numbers indicate the size of the DNA standards in kilobase pairs (Lane a).

Discussion
In our previous study, we validated the strong promoter activity from the pepR gene which encodes a DeoR family transcriptional regulator [12].The pepR promoter is activated by Sigma E and can direct cry1Ac expression efficiently [12].However, the functions of the pepR gene and its targets were unknown.In this study, we found that PepR homologs are widely distributed in various Gram-positive bacteria, and the amino acid similarities are highly conserved in different bacteria (Figure 1).We selected 18 strains that have been extensively studied for exhaustive analyses of the pepR genetic loci.We found that pepV, pepR and rusA-ytgP were closely linked in some strains, such as B. thuringiensis HD73, B. cereus ATCC 14579, B. cytotoxicus, B. anthracis Ames, Planococcus antarcticus, Ectobacillus sp.JY-23, Sporosarcina psychrophile, and Sutcliffiella horikoshii, while in some other strains they were not closely linked, such as in Anoxybacillus caldiproteolyticus, A. flavithermus, Priestia koreensis, Mangrovibacillus cuniculi, P. megaterium QM B1551, B. pumilus, Lysinibacillus fusiformis, Aeribacillus pallidus KCTC 3564, Metabacillus sediminilitoris, and Cytobacillus spongiae (Figure S1).In HD73, pepV and rusA-ytgP were two independent operons, and their promoters failed to transcript in the pepR knockout strain (Figures 2 and 3), suggesting these genes are all controlled by transcriptional regulator PepR.We further found that PepR directly binds to the pepV promoter but not to the rusA-ytgP promoter (Figures 4 and 5), revealing that pepV acts as a downstream target of pepR.The pepV promoter regions were 98.95% similar in different B. thuringiensis strains, and we determined that the expression of pepV is regulated by PepR.Whether PepR homologs also regulate pepV transcription in other bacteria remains to be confirmed.In addition, other targets of PepR should be investigated using RNA-seq and ChIP-seq methods in following studies.
PepV is highly conserved within the dipeptidase M20 family in the metallopeptidases [30].It has been reported that the active sites and conserved domains of all PepVs are similar [30][31][32].We analyzed the amino acid sequence of the PepV homologs and found that the identity varies in B. subtilis (57.36%),L. delbrueckii (34.62%),L. helveticus (33.12%), S. gordonii (40.86%) and L. lactis (38.92%) compared to HD73 (Figure S2).This result suggests that PepV in HD73 may have a similar function in catalyzing substrates to that reported in other bacteria.It is worth mentioning that the deletion of pepV in HD73 increased the susceptibility to vancomycin, whereas the deletion of pepV in L. lactis MG1363 decreased the susceptibility of L. lactis to vancomycin [29].In addition to BCARR regulating pepV expression in the presence of BCAAs in L. helveticus [17] and a CodY-like regulatory system controlling the expression of pepV in L. helveticus CM4 [16], pepV might have multiple functions in responding to different signals in different bacteria strains.

13 Figure 1 .
Figure 1.Evolution of PepR homologs in bacteria.Evolutionary analyses were conducted in MEGA7 using the neighbor-joining method.A total of 143 amino acid sequences were analyzed and an optimal tree was shown.Red text indicates the B. thuringiensis HD73 strain from which the reference sequence of the PepR homologs originated.

Figure 1 .
Figure 1.Evolution of PepR homologs in bacteria.Evolutionary analyses were conducted in MEGA7 using the neighbor-joining method.A total of 143 amino acid sequences were analyzed and an optimal tree was shown.Red text indicates the B. thuringiensis HD73 strain from which the reference sequence of the PepR homologs originated.

13 Figure 2 .
Figure 2. RT-PCR identified the pepV and rsuA-ytgP transcription units at the pepR gene locus in B. thuringiensis HD73.(A) Genetic organization of the pepR locus.Open reading frames (ORFs) are represented by grey arrows.The RT-PCR amplicons (see lanes in panel (B)) correspond to the dashed lines with small black arrows.The solid lines below the ORFs represent the operons.(B) RT-PCR analyzed the transcription units of pepR neighboring genes.RNA samples were prepared at T6 of bacterial culture in SSM medium.The RT-PCR labelled "C" was performed with 500 ng RNA.Positive controls are labelled "+" (PCR with 100 ng of genomic DNA).Negative controls are labelled "−" (RT-PCR with 500 ng RNA using heat-inactivated reverse transcriptase).Numbers represent different RT-PCR amplicons: numbers 1-4 represent thpR, pepV, rsuA, and ytgP; numbers 5 and 6 represent thpR-pepV and rsuA-ytgP, respectively.

Figure 2 .
Figure 2. RT-PCR identified the pepV and rsuA-ytgP transcription units at the pepR gene locus in B. thuringiensis HD73.(A) Genetic organization of the pepR locus.Open reading frames (ORFs) are represented by grey arrows.The RT-PCR amplicons (see lanes in panel (B)) correspond to the dashed lines with small black arrows.The solid lines below the ORFs represent the operons.(B) RT-PCR analyzed the transcription units of pepR neighboring genes.RNA samples were prepared at T 6 of bacterial culture in SSM medium.The RT-PCR labelled "C" was performed with 500 ng RNA.Positive controls are labelled "+" (PCR with 100 ng of genomic DNA).Negative controls are labelled "−" (RT-PCR with 500 ng RNA using heat-inactivated reverse transcriptase).Numbers represent different RT-PCR amplicons: numbers 1-4 represent thpR, pepV, rsuA, and ytgP; numbers 5 and 6 represent thpR-pepV and rsuA-ytgP, respectively.

Figure 3 .
Figure 3. Transcription of pepV and rsuA-ygtP depended on pepR.(A) Construction of an in-frame deletion pepR mutant in HD73.The pepR gene was substituted by a kanamycin-resistant gene through double homolog recombination events (B) Identification of ΔpepR knockout mutant by PCR.PCR products were amplified from ΔpepR (Lane b) and HD73 (Lane c) with primer pair pepR-WF/pepR-WR.To confirm the presence of the pMADΩDpepR plasmid in the ΔpepR genome, PCR was performed using pMAD plasmid universal primers (pMAD-F/pMAD-R) (Lane d).The numbers indicate the size of the DNA standards in kilobase pairs (Lane a).(C) β-galactosidase activity of PpepV-lacZ in HD73 and ΔpepR.(D) β-galactosidase activity of PrsuA-ygtP-lacZ in HD73 and ΔpepR.The promoters of pepV and rsuA-ygtP were fused with the lacZ reporter and transformed into HD73 and ΔpepR, respectively.The β-galactosidase activities of three clones were determined at the indicated times after growing the cells in SSM at 30 °C.Each value represents the mean and standard error of at least three independent replicates.

Figure 3 .
Figure 3. Transcription of pepV and rsuA-ygtP depended on pepR.(A) Construction of an in-frame deletion pepR mutant in HD73.The pepR gene was substituted by a kanamycin-resistant gene through double homolog recombination events (B) Identification of ∆pepR knockout mutant by PCR.PCR products were amplified from ∆pepR (Lane b) and HD73 (Lane c) with primer pair pepR-WF/pepR-WR.To confirm the presence of the pMADΩDpepR plasmid in the ∆pepR genome, PCR was performed using pMAD plasmid universal primers (pMAD-F/pMAD-R) (Lane d).The numbers indicate the size of the DNA standards in kilobase pairs (Lane a).(C) β-galactosidase activity of PpepV-lacZ in HD73 and ∆pepR.(D) β-galactosidase activity of PrsuA-ygtP-lacZ in HD73 and ∆pepR.The promoters of pepV and rsuA-ygtP were fused with the lacZ reporter and transformed into HD73 and ∆pepR, respectively.The β-galactosidase activities of three clones were determined at the indicated times after growing the cells in SSM at 30 • C. Each value represents the mean and standard error of at least three independent replicates.

Figure 5 .
Figure 5. Construction of an in-frame deletion pepV mutant in HD73.(A) Construction of an inframe deletion pepV mutant.The pepV gene was substituted by a kanamycin-resistant gene through double homolog recombination events.(B) Identification of the ΔpepR knockout mutant by PCR.PCR products were amplified from HD73 and ΔpepV strains using the primer pairs of pepV-WF/pepV-WR (Lanes b and c) and pepV-WF/pepV-KR (Lanes e and f).To confirm the presence of the pMADΩDpepV plasmid in the ΔpepV genome, PCR was performed using pMAD plasmid universal primers (pMAD-F/pMAD-R) (Lane d).The numbers indicate the size of the DNA standards in kilobase pairs (Lane a).

Figure 5 .
Figure 5. Construction of an in-frame deletion pepV mutant in HD73.(A) Construction of an in-frame deletion pepV mutant.The pepV gene was substituted by a kanamycin-resistant gene through double homolog recombination events.(B) Identification of the ∆pepR knockout mutant by PCR.PCR products were amplified from HD73 and ∆pepV strains using the primer pairs of pepV-WF/pepV-WR (Lanes b and c) and pepV-WF/pepV-KR (Lanes e and f).To confirm the presence of the pMADΩDpepV plasmid in the ∆pepV genome, PCR was performed using pMAD plasmid universal primers (pMAD-F/pMAD-R) (Lane d).The numbers indicate the size of the DNA standards in kilobase pairs (Lane a).

Figure 6 .
Figure 6.∆pepR and ∆pepV mutant strains were more sensitive to vancomycin compared to HD73.Bacteria growth was monitored every hour after inoculation to culture medium with vancomycin at 6 ng/mL.Growth curves represent means of three clones.

Table 1 .
Strains and plasmids used in this study.
a Antibiotic resistance cassettes are indicated as follows: Erm R , erythromycin resistance; Kan R , kanamycin resistance; Amp R , ampicillin resistance.

Table 2 .
Oligonucleotide primers used in this study.