Characterization of a novel cell wall hydrolase CwlE involved in Bacillus thuringiensis subsp. israelensis mother cell lysis

Cell wall hydrolases are ubiquitous among spore-form bacteria and essential for mother cell lysis. In this study, a novel cell wall hydrolase gene cwlE involved in mother cell lysis was characterized from Bacillus thuringiensis subsp. israelensis (Bti) strain Bt-59. cwlE was specifically expressed in Bti and located in the large plasmid carrying the insecticidal genes. The encoded CwlE protein consists of a MurNAc-LAA domain and two highly conserved catalytic residues (E26 and E151). The recombinant CwlE-His protein was able to digest the cell wall of Bti, indicating that CwlE is an N-acetylmuramoyl-L-alanine amidase. Transcriptional analysis indicated that cwlE began to express at the early stage of stationary phase and was controlled by SigE. Single mutation of cwlE gene delayed Bti mother cell lysis, while double mutation of cwlE and sigK completely blocked Bti mother cell lysis. After exposure to UV light to deactivate the crystal proteins, the level of decrease of insecticidal activity against mosquito larvae of Bt-59 (ΔcwlE-sigK) was less than that observed for Bt-59. This study elucidates the mechanism of Bti mother cell lysis and provides an effective strategy for mosquito control using Bt products with increased persistence.


Introduction
Mosquitoes are tropical disease vectors for transmitting malaria, dengue, filariasis, and yellow fever, posing an enormous public health menace (Reisen, 2010;Zhang et al., 2017).Although chemical pesticides are the most widespread way to control mosquitoes, they are losing the advantage due to pest resistance and environmental pollution (Maciel-de-Freitas et al., 2014;Omotayo et al., 2022).Bacillus thuringiensis subsp.israelensis (Bti) is a gram-positive bacterium that forms a parasporal crystal composed of protein protoxins with specific insecticidal activity against dipteran larvae (Federici et al., 2013;Ben-Dov, 2014).Long-term exposure of mosquito larvae to Bti did not evolve into resistance to this microbial larvicide, which supports its sustainable use for integrated control practices (Carvalho et al., 2018).The low probability of development of resistance to Bti is attributed to the synergistic interaction among its four main Cry toxins, namely Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1A (Vasquez et al., 2009).However, the main disadvantage of Bti is its short efficacy duration against mosquito larvae, limiting its widespread use as a larvicide.
Like other B. thuringiensis (Bt) strains, the durable spore and the parasporal crystal are set free with Bti mother cell lysis at the late sporulation stage (Ben-Dov, 2014).Then, the exposed insecticidal crystal proteins (ICPs) are easily inactivated by environmental factors, especially solar irradiation (Ramirez-Lepe et al., 2003;Zogo et al., 2019).An asporogenic Bt strain that expressed a chimeric cry1C/Ab gene showed high potency against various lepidopteran pests, and parasporal crystals produced by the strain remained encapsulated within the mother cells, which protected them from UV light degradation (Sanchis et al., 1999).Therefore, it is feasible to achieve the protection of ICPs from solar inactivation by knocking down the crucial cell wall hydrolase gene to block Bti mother cell lysis.
Three cell wall hydrolase genes (cwlB, cwlC, and cwlH) were identified in Bacillus subtilis, a well-established model for mother cell lysis (Vollmer et al., 2008).Except for the expression of cwlB in vegetative growth stage, cwlC and cwlH were expressed during the sporulation phase (Foster, 1992;Kuroda et al., 1993;Nugroho et al., 1999).Deleting of a single hydrolase gene did not affect mother cell lysis, but both cwlB-cwlc and cwlB-cwlH mutations blocked mother cell lysis of B. subtilis (Nugroho et al., 1999).While in B. thuringiensis subsp.kurstaki (Btk) strain HD73, the deletion of cwlC completely blocked Btk mother cell lysis, which lead to ICPs encapsulated in the mother cell (Chen et al., 2018).Based on the previous study, although CwlC of Bti showed 97% amino acid sequence identity with that of Btk, the deletion of cwlC just delayed the lysis of mother cells in Bti (Huang et al., 2022).The role of CwlC from Bti performed during the mother cell lysis was quite different from that of Btk, indicating that the hydrolysis of Bti mother cell wall involves a complex lysase system.
In the previous study, three cell wall hydrolase genes (cwlB, cwlC, and cwlE) were identified in the genome of Bti strain Bt-59 (Huang et al., 2022).cwlB and cwlC were regulated by SigK, a critical regulatory factor in the sporulation phase regulating genes involved in the formation of the outer wall of the spore, mother cell lysis, and spore germination (Zheng and Losick, 1990;Nugroho et al., 1999;Huang et al., 2022).The sigK and cwlC mutation strains of Bti were constructed in the previous study.As the key Sigma factor in regulating spore maturation and germination, the absence of sigK inhibits spore formation in Bti (Peng et al., 2016;Xu et al., 2020).Due to the major insecticidal genes of Bti are controlled by the sporulationspecific sigma factor SigE, the deletion of sigK did not have a significant impact on the production of ICPs in Bt-59 (Deng et al., 2014;Xu et al., 2020).In addition, the deletion of sigK or cwlC just delayed but not completely blocked Bti mother cell lysis (Xu et al., 2020;Huang et al., 2022).Moreover, cwlE was the only hydrolase gene with high expression level in the sigK mutant of Bti, indicating that cwlE was not controlled by SigK and CwlE was a critical autolysin leading to Bti mother cells lysis (Huang et al., 2022).Based on these studies, the double mutation of cwlE and sigK is likely to block Bti mother cell lysis without affecting its gene expression of ICPs.
This work analyzed the function and transcriptional regulation of cwlE, a new cell wall hydrolase gene of Bti.The cwlE mutation strain was constructed and the deletion of cwlE delayed Bti mother cell lysis.In addition, an ICPs encapsulation strain of Bti was constructed by deleting both sigK and cwlE.The result of insecticidal activity analysis showed that the sigK-cwlE mutant strain had better tolerance to ultraviolet than the Bti wild strain.Based on the characteristics analysis of CwlE and its role in mother cell lysis, we elucidated the mechanism of Bti mother cell lysis and constructed an ICPs encapsulation Bti strain with the improvement of insecticidal persistence.

DNA manipulation
Primers used in this study were synthesized in GENERAL BIOL (Hefei, China) and showed in Supplementary Table S1.PCR was performed using PrimeSTAR HS DNA polymerase (TaKaRa, Beijing, China) with corresponding primers.DNA fragments were purified with the TIANgel Midi Purification Kit (TIANGEN, Beijing, China) and plasmids were purified with the TIANprep Mini Plasmid Kit (TIANGEN, Beijing, China).The ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China) was used to clone the DNA fragments into the linearized vectors.Plasmids were introduced into the Bt cells by electroporation as previously described (Huang et al., 2022).

Strain construction
The temperature-sensitive suicide plasmid pMAD with the cwlE mutation sequence was constructed to delete the cwlE gene in the Bt-59 genome (Supplementary Figure S1).A 908-bp fragment upstream of cwlE (fragment A) was amplified by PCR, and cwlE-1 and cwlE-2 were used as primers.A 1013-bp fragment downstream of cwlE (fragment B) was amplified by PCR, and cwlE-3 and cwlE-4 were used as primers.The cwlE mutation box was amplified using fragments A and B as templates and cwlE-1 and cwlE-4 as primers by overlapping PCR.The resulting fragment was cloned into the pMAD plasmid to generate pMADΔcwlE plasmid.Then, the recombinant plasmid pMADΔcwlE was transformed into Bt-59 and Bt-59 (ΔsigK) to generate the cwlE deletion mutant Bt-59 (ΔcwlE) and the sigK and cwlE double mutant .

Expression and cell wall hydrolysis of recombinant CwlE protein
After strains BL21(pETcwlE), BL21(pETcwlC-E24A), and BL21(pETcwlC-E151A) were grown in LB liquid medium (Amp, 100 μg/mL) at 37°C for 4 h with shaking at 220 rpm, 0.5 mM isopropyl-beta-D-thiogalactoside (Sangon, Shanghai, China) was added.The bacteria were cultured at 16°C for another 14 h with shaking at 160 rpm to induce protein expression.Then, the recombinant CwlE proteins with a His tag were purified according to the method described previously (Zhang et al., 2020).After grown in LB liquid medium at 30°C for 6 h with shaking at 220 rpm, Bt-59 was collected by centrifugation at 10000 rpm.Then, the bacteria were resuspended with TK buffer (0.05 M Tris-HCl, 0.05 M KCl, pH 7.0) and disrupted with a FastPrep-24 (MPBiomedicals).Unbroken cells were removed by low-speed centrifugation (3,500 rpm, 5 min).The crude cell walls were pelleted at 13500 rpm for 5 min at 4°C.Finally, the pellets were resuspended in TK buffer.The purified CwlE proteins were added to the prepared cell wall of Bt-59 for hydrolytic activity analysis (Huang et al., 2022).After incubated at 37°C, the OD 540 of the mixtures were measured at the designated time point (Nugroho et al., 1999).

Analysis of the transcription start site
Total RNA was extracted from Bt-59 cultured in SSM at T 15 with TRIzol reagent (Invitrogen, USA).The transcription start site of the cwlE gene was determined with 5' RACE according to the manufacturer's instruction of HiScript-TS 5′/3' RACE Kit (Vazyme, Nanjing, China).The primers used for 5' RACE are listed in Supplementary Table S1.

β-Galactosidase assays
Bt strains HD (P cwlE -lacZ) and HD (ΔsigE) (P cwlE -lacZ) were cultured in SSM (Ery, 25 μg/mL) at 30°C with shaking at 220 rpm.Two-milliliter bacterial suspensions of HD (P cwlE -lacZ) and HD (ΔsigE) (P cwlE -lacZ) were collected from T 0 to T 20 at 1-h intervals (T 0 is the end of the exponential phase and T n is n hours after the end of the exponential phase).The cells were harvested and the samples were prepared as previously described (Chen et al., 2018).Then, the specific β-galactosidase activity was measured and expressed as Miller units per milligram of protein (Chen et al., 2018).The values are the means for three independent experiments.

Quantitative real-time PCR (qPCR)
Total RNA was extracted from Bt strains cultured in SSM at T 0 , T 8 and T 16 with TRIzol reagent (Invitrogen, USA).T 0 is the end of the exponential phase and T n is n hours after the end of the exponential phase.The purified RNA was reverse transcribed into cDNA using HiScript IV RT SuperMix for qPCR (gDNA wiper) (Vazyme, Nanjing, China).RpsU and gatB were chosen as reference genes (Reiter et al., 2011).qPCR was performed using specific primers (Supplementary Table S1) and ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) with a 7,500 Fluorescence Quantitative PCR Instrument (ABI, California, USA).The specificity and qualified amplification efficiency of the specific primers were verified.Three biological replicates were designed.The relative expression level of each target gene was calculated according to the 2 -ΔCT method (Huggett et al., 2013).

Assays of vegetative growth and Cry proteins production
The wild-type strain Bt-59, cwlE deletion mutant Bt-59 (ΔcwlE), and double mutant Bt-59 (ΔcwlE-sigK) were cultured in SSM medium at 30°C with shaking at 220 rpm.The growth curves of Bti strains were drew by measuring the OD 600 values of the culture solutions every hour.After 12 h of cultivation, the number of colonies of Bt-59 and its mutants were calculated by the coating plate method.SDS-PAGE was used for the crystal protein production assay as described previously (Xu et al., 2020).

Microscopic analysis
Bt strains were cultured in SSM medium at 30°C with shaking at 220 rpm to the certain time points.Samples were collected and centrifuged, and then sediments were suspended with the appropriate volume of deionized water.Bacterial suspension was placed on a glass slide, and then cell morphology was observed by optical microscopy (ZEISS, Germany).

Bioassay of insecticidal activity
Bt-59 and Bt-59 (ΔcwlE-sigK) were cultured in a fermentation medium at 30°C with shaking at 220 rpm for 44 h as described previously (Huang et al., 2022).The fermentation media were serially diluted with water to the appropriate concentrations and used for the bioassay.Twenty larvae of Culex pipiens and Aedes albopictus in the early fourth instar were collected for bioassay in each treatment (Huang et al., 2022).Then the fermentation media were exposure to 365 nm UV light for 30 min used for the bioassay (Patel et al., 1996).The experiment was performed with three replicates at 28°C in a biochemical incubator for 24 h.The distilled water was used as a control.

Sequence and data analysis
Vector NTI 11 and GENEDOC were used for sequence analysis.The primer design was performed by Primer Premier 5.0.The phylogentic tree was constructed with MEGA Microsoft Office Excel and GraphPad Prism 6 were used for data analysis.PoloPlus statistical software was used for estimating the LC 50 values and 95% confidence intervals (CIs) (Straus, 2008).Lethal dose ratios (LC 50 ratio, LCR) were calculated to compare natural variation between different treatments, and the LCR value was considered to be significant (p < 0.05) when the confidence intervals did not encompass the value 1 (Straus, 2008).

Bioinformatic analysis of CwlE
To identify the essential cell wall hydrolase in Bti mother cell lysis, a novel hydrolase gene named cwlE was identified from a 128 kb sized plasmid (CP039725.1)encoding multiple insecticidal protein genes of the strain Bt-59.cwlE is 816 base pairs in length and encodes a putative cell wall hydrolase with the MurNAc-LAA domain and two highly conserved catalytic residues E26 and E151 (Figure 1).The amino acid sequence identity showed CwlE shares 32% of its amino acid sequence identity with CwlM of B. licheniformis, 28% with CwlC of Bt-59, and 33% with CwlC of B. subtilis.The absence of a homologous sequence of cwlE in the genomes of bacteria other than Bti strains, as determined by the NCBI homology analysis, suggests that cwlE is a cell wall hydrolase gene uniquely expressed in Bti.

Hydrolytic activity against cell wall of CwlE
To investigate the function of CwlE in Bti, the recombinant CwlE-His protein was purified using nickel column affinity chromatography.To determine whether the conserved glutamic acids are essential for enzymatic catalytic activity, two distinct single amino acid substitutions (E26A and E151A) were separately introduced into the CwlE-expressing strains using site-directed mutagenesis.SDS-PAGE showed that the molecular weight of the recombinant CwlE and its mutant proteins is approximately 35 kDa (Figure 2A).Then, mixtures of the recombinant proteins and the Bti cell wall were incubated at 37°C to determine the cell wall lytic ability of the CwlE and its mutant proteins.After incubation of CwlE with the cell wall for 60 min, the optical density (OD) of the mixture decreased by about 24% (Figure 2B).In contrast to the wild-type CwlE protein, the two mutant CwlE proteins (E26A and E151A) exhibited no catalytic activity against the Bti cell wall (Figure 2B).
After 15 days of growth in SSM medium the mother cells of Btk mutant strain HD (ΔcwlC) did not autolyze in a previous study (Chen et al., 2018).The cwlE gene containing its promoter region sequence was transferred into HD (ΔcwlC) strain to further determine the cell wall lytic ability of CwlE.Cells of HD (ΔcwlC::cwlE) began to autolyze at T 22 and completely autolyzed after 8 h of continuous culture (Figure 2C).The result indicated that the promoter of cwlE gene showed transcriptional activity in Btk, and CwlE could hydrolyze cell wall of HD73 and restore the phenotype of HD (ΔcwlC).

Transcriptional regulation of the cwlE gene
The result of 5' RACE-PCR experiment showed that the transcription start site of cwlE gene is a T located 97 nucleotides upstream of its translational start codon ATG (Figure 3A).The putative cwlE promoter contains the sequences TAATATCT and AATAAAAT, which are located −35 and − 10 nucleotides upstream of the cwlE transcription start site, respectively (Figure 3A).These sequences are similar to those in the transcriptional regulatory factor SigE dependent promoters (KHATANHT and MATANNHT; H is A, C, or T; K is G or T; M is A or C; N is A, C, G, or T.) (Du et al., 2012), suggested that the cwlE promoter might be recognized and the expression of the cwlE gene was regulated by SigE.
The results of qPCR showed that the cwlE gene containing its promoter could be expressed in HD73, while it was almost undetectable in HD (ΔsigE) (Figure 3B).To further determine  whether cwlE is regulated in a SigE-dependent manner, the Bt-59 (P cwlE -lacZ), HD (P cwlE -lacZ), and HD (ΔsigE) (P cwlE -lacZ) strains were constructed.β-Galactosidase activity assays showed that reporter vector was expressed both in Bt-59 and HD73, but not in sigE mutant strain of HD73 (Figure 3C).The result showed that the cwlE promoter could be recognized by SigE in Bti and Btk, and the expression of cwlE was controlled by SigE.In Bt-59, the expression of the reporter vector started at T 0 , reached a maximum at T 13 , and then decreased in the following time (Figure 3C).Furthermore, the transcriptional activity of the cwlE promoter did not differ significantly between Bt-59 and HD73 (Figure 3C).
The expression levels of four main insecticidal protein genes (cry4Aa, cry4Ba, cry11Aa, and cyt1Aa) in Bt-59, Bt-59 (ΔcwlE), Bt-59 (ΔsigK), and Bt-59 (ΔcwlE-sigK) were revealed by qPCR (Figure 5).Compared with the wild strain, the deletion of cwlE or sigK did not affect the expression of cry4Aa and cry4Ba at T 0 , T 8 , and T 16 .cry11Aa showed similar expression levels in the four strains at T 0 and T 8 , while its expression was reduced in the mutants Bt-59 (ΔsigK) and Bt-59 (ΔcwlE-sigK) at T 16 .The deletion of cwlE did not affect the expression of cyt1Aa, while the expression of cyt1Aa was reduced in cwlE and sigK double mutation strain Bt-59 (ΔcwlE-sigK).Besides, Cry proteins production of Bt-59 and its mutants were examined by SDS-PAGE after being cultured in SSM medium for 24 h.The results showed that deletion of cwlE had no effect on Cry proteins production in Bti, while the cwlE-sigK deletion decreased the expression of Cry11Aa (Supplementary Figure S3).

Deletion of the cwlE gene delayed mother cell lysis
Optical microscopy was used to investigate the morphology of the Bt-59 and Bt-59 (ΔcwlE) grown in SSM medium to various growth phases (Figure 6).The result showed that cells of Bt-59 began to lyse at T 16 , while cells of Bt-59 (ΔcwlE) began to lyse at T 20 .Up to T 24 , the cells of Bt-59 were completely lysed, and the most of the cells of Bt-59 (ΔcwlE) were lysed.After another 4 hours of culture, the cells of Bt-59 (ΔcwlE) were completely lysed.In addition, the cells of cwlE gene restoration strain Bt-59 (ΔcwlE::cwlE) began to autolyze at T 16 , and virtually all of the cells were lysed at T 24 , which was similar to Bt-59.

cwlE and sigK double mutant completely blocked mother cell lysis
To further verify the role of cwlE in mother cell lysis of Bti, the cell wall hydrolase gene cwlE and the transcription regulatory factor gene sigK double deletion mutant Bt-59 (ΔcwlE-sigK) was constructed based on the sigK mutant strain.Optical microscopy was used to examine the morphology of Bt-59 (ΔsigK) and Bt-59 (ΔcwlE-sigK) grown in SSM medium to various growth phases (Figure 7A).The result showed that parts of cells of Bt-59 (ΔsigK) were lysed at T 48 .Up to T 72 , the cells of Bt-59 (ΔsigK) were completely lysed, whereas mother cell lysis of Bt-59 (ΔcwlE-sigK) could not be observed.The cell morphology of the complemented strain Bt-59 (ΔcwlE-sigK::cwlE) was comparable to that of the sigK mutant strain.Furthermore, Bt-59 (ΔcwlE-sigK) was cultured in SSM for 7 days to further examine the mother cell lysis phenotype.The results showed that the cells of Bt-59 (ΔcwlE-sigK) still did not autolyze after 7 days of growth (Figure 7B).

Insecticidal activity against mosquito larvae
The early fourth-instar larvae of Culex pipiens and Aedes albopictus were fed with fermentation suspension serially diluted with water to the appropriate concentrations to determine the insecticidal activity of the cwlE and sigK double deletion mutant Bt-59 (ΔcwlE-sigK).The LC 50 values of Bt-59 and Bt-59 (ΔcwlE-sigK) against C. pipiens were 0.314 μL/L and 0.364 μL/L, respectively (Table 2).After exposure to UV light, the insecticidal activity of Bt-59 and Bt-59 (ΔcwlE-sigK) against C. pipiens reduced by 86.62 and 2.47%, respectively (Table 2).The LC 50 values of Bt-59 and Bt-59 (ΔcwlE-sigK) against A. albopictus were 2.066 μL/L and 2.075 μL/L, respectively (Table 3).After exposure to UV light, the insecticidal activity of Bt-59 and Bt-59 (ΔcwlE-sigK) against A. albopictus reduced by 52.76 and 12.43%, respectively (Table 3).The hypothesis of equality and parallelism showed that the cwlE and sigK double mutant did not affect the insecticidal activity of Bti against mosquito larvae (Figures 8A,C).In contrast, the level of decrease of insecticidal activity against mosquito larvae of Bt-59 (ΔcwlE-sigK) was less than that observed for Bt-59 after UV treatment (Figures 8B,D).

Discussion
N-acetylmuramyl-L -alanine amidase contains the MurNAc-LAA domain and hydrolyzes the β-1,4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid in polysaccharide chain, which ubiquitous among bacteria and essential for the mother cell lysis (Vollmer et al., 2008;Bhagwat et al., 2020).
CwlC is an essential cell wall hydrolase in Btk mother cell lysis and was distributed among the members of the B. cereus group (Chen et al., 2018).The CwlC exhibited the identical critical catalytic residues and conservative domains as Btk, whereas the deletion of cwlC delayed the lysis of mother cells in Bti (Huang et al., 2022).Although the mutation of sigK could inhibit the expression of the cell wall hydrolase genes cwlC and cwlB, it could not block cell wall lysis in Bti (Xu et al., 2020;Huang et al., 2022).This evidence demonstrates conclusively  that Bti additional crucial cell wall hydrolases associated with mother cell lysis.Bti's novel cell wall hydrolase gene, cwlE, which is located on the large plasmid carrying the insecticidal genes, was characterized in this study.The absence of a cwlE homolog in other bacterial genomes, as determined by the NCBI homology analysis, indicates that cwlE is exclusively expressed in Bti.The CwlE is a new N-acetylmuramyl-Lalanine amidase, consisting of a MurNAc-LAA domain and two highly conserved catalytic residues (E26 and E151).Similar to the cwlC deletion mutant, the cwlE deletion mutant was unable to entirely prevent Bti mother cell lysis.In B. subtillis, cell wall lytic enzymes were functionally redundant because none of the single autolysin gene The expression levels of cry4Aa, cry4Ba, cry11Aa, and cyt1Aa in Bt-59, Bt-59 (ΔcwlE), Bt-59 (ΔsigK), and Bt-59 (ΔcwlE-sigK) at T 0 , T 8 , and T 16 were revealed by qPCR.Each value represents the mean of at least three independent replicates.Error bars represent the standard error of the mean.T 0 is the end of the exponential phase and T n is n hours after the end of the exponential phase.Statistically significant difference analysis was performed by ordinary one-way ANOVA (p < 0.05). 10.3389/fmicb.2023.1250542 Frontiers in Microbiology 10 frontiersin.orgdeletions affected cell lysis and Foster, 1995;Nugroho et al., 1999).However, the single mutant of autolysin genes delayed mother cell lysis in Bti, which differed from that in B. subtillis.Sigma factors influence physiological process by regulating the transcription of various peptidoglycan hydrolase genes at various growth phases in Bacillus (Smith et al., 2000).Until now, the cell wall hydrolases involved in mother cell lysis were regulated by SigK in Bt strains (Yang et al., 2013;Chen et al., 2018;Huang et al., 2022).Analysis of promoter transcription activity showed that cwlE was regulated by SigE, which is the key Sigma factor regulating the expression of major insecticidal genes during the early sporulation stage in Bti (Deng et al., 2014).Our finding indicated that CwlE might be the key cell wall hydrolase causing the mother cell lysis in the sigK deletion mutant.To further verify the role of CwlE in Bti mother cell lysis, a double mutant strain was constructed by deleting of cwlE and sigK in this study.The results showed that deletion of cwlE and sigK could completely block Bti mother cell lysis, and cell wall lysis of double mutant could not be observed even after 7 days of culture in SSM medium.Therefore, we hypothesized that the cell wall lysis of Bti was accomplished through the synergy of the three hydrolases, CwlC, CwlB, and CwlE, and the absence of any cell wall hydrolases could delay mother cell lysis.
When Bt formulation is applied in the field, ultraviolet radiation is a key factor leading to crystal protein inactivation and reducing its Effect of cwlE deletion on mother cell lysis observed by optical microscopy.Mother cell lysis of Bt-59 and Bt-59 (ΔcwlE::cwlE) was observed at T 16 , T 20 , and T 24 .Mother cell lysis of mutant strain Bt-59 (ΔcwlE) was observed at T 16 , T 20 , T 24 and T 28 .T 0 is the end of the exponential phase and T n is n hours after the end of the exponential phase.Green arrows, red arrows, and blue arrows represent vegetative cells, spores, and crystal proteins, respectively.Scale bars, 5 μm., 2011).In the previous study, the mature crystal was produced and encapsulated in mother cells of a sigK deletion mutant of Bt strain, resulting in increased insecticidal activity against lepidoptera pests and greater resistance against UV radiation (Sanchis et al., 1999).In our study, the double mutant strain Bt-59 (ΔcwlE-sigK) showed increased insecticidal activity against the mosquito larva than the wild-type strain after exposure to UV light.
By blocking mother cell lysis, it is possible to engineer a Bt strain with enhanced insecticidal persistence according to the current research.We elucidated the function of CwlE in Bti mother cell lysis by deleting the hydrolase gene cwlE in this study.However, due to the lack of experimental data, we are not yet clear about the physiological function of cwlE expression at the early stage of stationary phase.Furthermore, compared to Btk, the reason why Bti  requires more cell wall hydrolases to participate in mother cell lysis is also unclear.We speculate that due to the different midgut environments of different target insects, the synergistic effect of multiple cell wall hydrolases may allow Bti to release spores and crystal proteins faster in the mosquito midgut to complete population reproduction.Further investigations are needed to confirm this hypothesis.

Conclusion
cwlE is a novel N-acetylmuramyl-L -alanine amidase gene in Bti strain Bt-59 and its expression is dependent on transcriptional regulation factor SigE. Deletion of cwlE delayed Bti mother cell lysis mother without impacting the vegetative growth and Cry proteins production.As a cell wall hydrolase uniquely expressed in Bti, CwlE is the key hydrolase causing the mother cell lysis in the sigK deletion mutant.The double mutation of cwlE and sigK completely blocked Bti mother cell lysis even after 7 days of growth.Due to the impact of the double mutation on the expression level of the major crystal protein genes were not significant, Bt-59 (ΔcwlE-sigK) still maintains high insecticidal activity against mosquito larvae.In addition, the level of decrease of insecticidal activity against mosquito larvae of Bt-59 (ΔcwlE-sigK) was less than that observed for Bt-59 after UV treatment.In summary, this study identified a novel and unique cell wall hydrolase gene cwlE, elucidated the mechanism of Bti mother cell lysis, and provided an effective strategy for mosquito control using crystal encapsulation of Bt strain with the increased resistance against UV light inactivation.

FIGURE 2
FIGURE 2 Expression of CwlE and its hydrolytic activity of cell wall.(A) SDS-PAGE analysis of recombinant CwlE and its mutant proteins purified by nickel column affinity chromatography.M, protein molecular size marker.CwlE, wild-type CwlE protein; E26A, replacement of glutamic acid with alanine of CwlE protein at position 24; E151A, replacement of glutamic acid with alanine of CwlE protein at position 151.(B) Digestion of Bacillus thuringiensis subsp.israelensis cell walls by purified CwlE and its mutant proteins.Cell walls incubated with addition of BSA protein were used as control.Each value represents the mean of at least three independent replicates.Error bars show standard deviations.(C) Mother cell lysis of HD (ΔcwlC::cwlE) was observed by optical microscopy after treatment with biological staining agent basic red at T 18 , T 22 , T 26 , and T 30 .T 0 is the end of the exponential phase and T n is n hours after the end of the exponential phase.Green arrows, red arrows, and blue arrows represent vegetative cells, spores, and crystal proteins, respectively.Scale bars, 5 μm.

FIGURE 3
FIGURE 3 Analysis of promoter sequence and transcriptional regulation of the cwlE gene.(A) Sequence analysis of the promoter regions from Bt-59 cwlE.The predicted transcription start site T is denoted with arrow, while the putative −35 and − 10 motifs are underlined and denoted.The translation start codon and ending codon of cwlE are marked shaded in red.(B) Expression level of cwlE gene in HD (cwlE) and HD (ΔsigE) (cwlE) at T 0 , T 8 , and T 16 .Each value represents the mean of at least three independent replicates.Error bars represent the standard error of the mean.T 0 is the end of the exponential phase and T n is n hours after the end of the exponential phase.(C) Assays of β-galactosidase activity of the cwlE promoter in Bt-59, HD73, and HD (ΔsigE).T 0 is the end of the exponential phase and T n is n hours after the end of the exponential phase.Each value represents the mean of at least three independent replicates.The error bars represent standard deviations.

FIGURE 4
FIGURE 4 Effect of the cwlE and cwlE-sigK deletion on the vegetative growth and Cry proteins production of Bt-59.(A) The cwlE deletion mutants were verified by qPCR.Each value represents the mean of at least three independent replicates.Error bars represent the standard error of the mean.T 0 is the end of the exponential phase and T n is n hours after the end of the exponential phase.(B) Growth curves of Bt-59, Bt-59 (ΔcwlE), and Bt-59 (ΔcwlE-sigK).Each value represents the mean of at least three independent replicates.The error bars represent standard deviations.(C) After culturing in SSM medium for 10 h, the number of Bt-59, Bt-59 (ΔcwlE), and Bt-59 (ΔcwlE-sigK) cells was detected by viable count.Each value represents the mean of at least three independent replicates.The error bars represent standard deviations.Statistically significant difference analysis was performed by ordinary one-way ANOVA (p < 0.05).

FIGURE 7
FIGURE 7 Effect of cwlE and sigK double deletion on mother cell lysis observed by optical microscopy.(A) Mother cell lysis of Bt-59 (ΔsigK), Bt-59 (ΔcwlE-sigK), and Bt-59 (ΔcwlE-sigK::cwlE) were observed at T 48 , T 60 , and T 72 .T 0 is the end of the exponential phase and T n is n hours after the end of the exponential phase.Green arrows and blue arrows represent vegetative cells and crystal proteins, respectively.Scale bars, 5 μm.(B) Mother cell lysis of mutant strain Bt-59 (ΔcwlE-sigK) was observed at 5 d, 6 d, and 7d.Green arrows represent vegetative cells.Scale bars, 5 μm.

TABLE 1
Strains and plasmids used in this study.

TABLE 2
Insecticidal activities of Bti strains against Culex pipiens.

TABLE 3
Insecticidal activities of Bti strains exposure to UV light against Aedes albopictus.