Cell death dependent on holins LrgAB repressed by a novel ArsR family regulator CdsR.

The cell death and survival paradox in various biological processes requires clarification. While spore development causes maternal cell death in Bacillus species, the involvement of other cell death pathways in sporulation remains unknown. Here, we identified a novel ArsR family transcriptional regulator, CdsR, and found that the deletion of its encoding gene cdsR causes cell lysis and inhibits sporulation. To our knowledge, this is the first report of an ArsR family transcriptional regulator governing cell death. We found that CdsR directly repressed lrgAB expression. Furthermore, lrgAB overexpression resulted in cell lysis without sporulation, akin to the cdsR mutant, suggesting that LrgAB, a holin-like protein, induces cell death in Bacillus spp. The lrgAB mutation increases abnormal cell numbers during spore development. In conclusion, we propose that a novel repressor is vital for inhibiting LrgAB-dependent cell lysis.

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.

Transcriptional regulation of cellobiose utilization by PRD-domain containing Sigma54-dependent transcriptional activator (CelR) and catabolite control protein A (CcpA) in Bacillus thuringiensis.

Cellobiose, a ß-1,4-linked glucose dimer, is a major cellodextrin resulting from the enzymatic hydrolysis of cellulose. It is a major source of carbon for soil bacteria. In bacteria, the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS), encoded by the cel operon, is responsible for the transport and utilization of cellobiose. In this study, we analyzed the transcription and regulation of the cel operon in Bacillus thuringiensis (Bt). The cel operon is composed of five genes forming one transcription unit. ß-Galactosidase assays revealed that cel operon transcription is induced by cellobiose, controlled by Sigma54, and positively regulated by CelR. The HTH-AAA+ domain of CelR recognized and specifically bound to three possible binding sites in the celA promoter region. CelR contains two PTS regulation domains (PRD1 and PRD2), which are separated by two PTS-like domains-the mannose transporter enzyme IIA component domain (EIIAMan) and the galactitol transporter enzyme IIB component domain (EIIBGat). Mutations of His-546 on the EIIAMan domain and Cys-682 on the EIIBGat domain resulted in decreased transcription of the cel operon, and mutations of His-839 on PRD2 increased transcription of the cel operon. Glucose repressed the transcription of the cel operon and catabolite control protein A (CcpA) positively regulated this process by binding the cel promoter. In the celABCDE and celR mutants, PTS activities were decreased, and cellobiose utilization was abolished, suggesting that the cel operon is essential for cellobiose utilization. Bt has been widely used as a biological pesticide. The metabolic properties of Bt are critical for fermentation. Nutrient utilization is also essential for the environmental adaptation of Bt. Glucose is the preferred energy source for many bacteria, and the presence of the phosphotransferase system allows bacteria to utilize other sugars in addition to glucose. Cellobiose utilization pathways have been of particular interest owing to their potential for developing alternative energy sources for bacteria. The data presented in this study improve our understanding of the transcription patterns of cel gene clusters. This will further help us to better understand how cellobiose is utilized for bacterial growth.

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.

Key amino acids residues enhance the ability of CpcR to activate cry gene expression in Bacillus thuringiensis.

Typical Bacillus thuringiensis (Bt) produces one or more parasporal crystals composed of insecticidal Cry proteins during the sporulation, and the parasporal crystals and spores are produced from the same cell. Strain Bt LM1212 is different from typical Bt strains in that its crystals and spores are produced in different cells. Previous studies have found that the cell differentiation process of Bt LM1212 is related to the transcription factor CpcR which activates the cry-gene promoters. In addition, CpcR could activate the Bt LM1212 cry35-like gene promoter (P35) when introduced in the heterologous HD73- strain. It was shown that P35 was only activated in non-sporulating cells. In this study, the peptidic sequences of CpcR homologous proteins found in other strains of the Bacillus cereus group were used as references to identify two key amino acid sites for CpcR activity. The function of these amino acids was investigated by measuring P35 activation by CpcR in strain HD73-. These results will lay a foundation for the optimization of the insecticidal protein expression system in non-sporulating cells.

Deletion of the novel gene mother cell lysis X results in Cry1Ac encapsulation in the Bacillus thuringiensis HD73.

The novel protein MclX (mother cell lysis X) in Bacillus thuringiensis subsp. kurstaki strain HD73 (B. thuringiensis HD73) was characterized in this work. MclX has no known domain and its gene deletion in HD73 resulted in Cry1Ac encapsulation in the mother cell and did not influence Cry1Ac protein production or insecticidal activity. In vitro cell wall hydrolysis experiments showed that MclX cannot hydrolyze the cell wall. In mclX deletion mutants, the expression of cwlC (which encodes a key cell wall hydrolase) was significantly decreased, as shown by the ß-galactosidase activity assay. MclX cannot directly bind to the cwlC promoter, based on the electrophoretic mobility shift assay (EMSA). The cwlC was reported to be regulated by σK and GerE. However, the transcriptional activities of sigK and gerE showed no difference between HD73 and the mclX deletion mutant. It is indicated that MclX influenced cwlC expression independently of σK or GerE, through a new pathway to regulate cwlC expression. mclX deletion could be a new approach for insecticidal protein encapsulation in Bacillus thuringiensis.

The Transcription Factor CpcR Determines Cell Fate by Modulating the Initiation of Sporulation in Bacillus thuringiensis.

Bacillus thuringiensis is a bacterium capable of differentiating into a spore, a dormant and highly resistant cellular form. During the sporulation process, this bacterium produces insecticidal toxins in the form of a crystal inclusion, usually in the sporulating cell. We previously reported that the B. thuringiensis LM1212 strain can differentiate into two distinct subpopulations of sporeformers and crystal producers and that this division-of-labor phenotype provides the bacterium with a fitness advantage in competition with a typical B. thuringiensis strain. The transcription factor CpcR was characterized as the regulator responsible for this phenotype. Here, we examined how CpcR interacts with the sporulation network to control the cell differentiation. We found that the sporulation process was inhibited prior to polar septum formation and that Spo0A activity was impaired in the presence of cpcR in strain LM1212. Using bioinformatics and genetic tools, we identified a gene positively controlled by CpcR encoding a putative phosphatase of the Spo0E family known to specifically dephosphorylate phosphorylated Spo0A (Spo0A-P). We showed that this protein (called Spo0E1) is a negative regulator of sporulation and that variations in spo0E1 expression can modulate the production of spores. Using fluorescent reporters to follow gene expression at the single-cell level, we correlated expression of cpcR and sporulation genes to the formation of the two differentiated subpopulations. IMPORTANCE Formation of spores is a paradigm for study of cell differentiation in prokaryotes. Sporulation initiation is governed by a gradual increase in the level and activity of the master regulator Spo0A. Spo0A is usually indirectly phosphorylated by a multicomponent phosphorelay, and modulation of this phosphorelay system is a critical aspect of Bacillus physiology. Though we know that this phosphorelay system is usually affected by two negative regulatory mechanisms, i.e., rap genes and spo0E family genes, the regulatory mechanisms controlling the transcription of these genes are poorly understood. Here, we report that the transcription factor CpcR positively regulates a spo0E family gene and that variations in spo0E expression can modulate the production of spores in B. thuringiensis. This work emphasizes the diversity in modes of sporulation and illustrates the diversity in the strategies employed by bacteria to control this differentiation pathway and ensure their survival.

Identification and Functional Characterization of Two Homologous SpoVS Proteins Involved in Sporulation of Bacillus thuringiensis.

Sporulation is an important part of the life cycle of Bacillus thuringiensis and the basis for the production of parasporal crystals. This study identifies and characterizes two homologous spoVS genes (spoVS1 and spoVS2) in B. thuringiensis, both of whose expression is dependent on the σH factor. The disruption of spoVS1 and spoVS2 resulted in defective B. thuringiensis sporulation. Similar to Bacillus subtilis, B. thuringiensis strain HD(ΔspoVS1) mutants showed delayed formation of the polar septa, decreased sporulation efficiency, and blocked spore release. Different from B. subtilis, B. thuringiensis HD(ΔspoVS1) mutants had disporic septa and failed to complete engulfment in some cells. Moreover, HD(ΔspoVS2) mutants had delayed spore release. The effect of spoVS1 deletion on polar septum delay and sporulation efficiency could be compensated by spoVS2. ß-Galactosidase activity analysis showed that the expression of pro-sigE and spoIIE decreased to different degrees in the HD(ΔspoVS1) and HD(ΔspoVS2) mutants. The different effects of the two mutations on the expression of sporulation genes led to decreases in Cry1Ac production of different levels. IMPORTANCE There is only one spoVS gene in B. subtilis, and its effects on sporulation have been reported. In this study, two homologous spoVS genes were found and identified in B. thuringiensis. The different effects on sporulation and parasporal crystal protein production in B. thuringiensis and their relationship were investigated. We found that these two homologous spoVS genes are highly conserved in the Bacillus cereus group, and therefore, the functional characterization of SpoVS is helpful to better understand the sporulation processes of members of the Bacillus cereus group.

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

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 ß-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.

Complete Genome Sequence and Function Gene Identify of Prometryne-Degrading Strain Pseudomonas sp. DY-1.

The genus Pseudomonas is widely recognized for its potential for environmental remediation and plant growth promotion. Pseudomonas sp. DY-1 was isolated from the agricultural soil contaminated five years by prometryne, it manifested an outstanding prometryne degradation efficiency and an untapped potential for plant resistance improvement. Thus, it is meaningful to comprehend the genetic background for strain DY-1. The whole genome sequence of this strain revealed a series of environment adaptive and plant beneficial genes which involved in environmental stress response, heavy metal or metalloid resistance, nitrate dissimilatory reduction, riboflavin synthesis, and iron acquisition. Detailed analyses presented the potential of strain DY-1 for degrading various organic compounds via a homogenized pathway or the protocatechuate and catechol branches of the ß-ketoadipate pathway. In addition, heterologous expression, and high efficiency liquid chromatography (HPLC) confirmed that prometryne could be oxidized by a Baeyer-Villiger monooxygenase (BVMO) encoded by a gene in the chromosome of strain DY-1. The result of gene knock-out suggested that the sulfate starvation-induced (SSI) genes in this strain might also involve in the process of prometryne degradation. These results would provide the molecular basis for the application of strain DY-1 in various fields and would contribute to the study of prometryne biodegradation mechanism as well.

Characteristics of the sigK Deletion Mutant from Bacillus thuringiensis var. israelensis Strain Bt-59.

All major insecticidal genes of Bacillus thuringiensis var. israelensis (Bti) are controlled by the sporulation-specific sigma factor Sigma E (sigE), while sigE is negatively regulated by Sigma K (sigK). Therefore, knocking out sigK plays an important role in regulating the expression of insecticidal genes in Bti. A sigK deletion mutant of B. thuringiensis var. israelensis strain Bt-59, Bt59(ΔsigK), was constructed by homologous recombination and characterized. The sigK deletion resulted in no mature spores and delayed mother cell lysis from T25 to T60, while the genetically complemented strain, Bt59(HFsigK), had mother cell lysis at T25. Compared to Bt-59, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis indicated that the expression of Cry4Aa2/4Ba1 and Cyt1Aa1 proteins in Bt59(ΔsigK) increased approximately 1.67 and 1.21 times, respectively. However, there was no significant change in Cry11Aa1 protein expression between the two strains. Bioassay results showed that the sigK deletion mutation slightly reduced the insecticidal activity of Bt-59 against Culex pipiens pallens and did not obviously affect activity against Aedes albopictus.

A Bacillus thuringiensis Chitin-Binding Protein is Involved in Insect Peritrophic Matrix Adhesion and Takes Part in the Infection Process.

Bacillus thuringiensis (Bt) is used for insect pest control, and its larvicidal activity is primarily attributed to Cry toxins. Other factors participate in infection, and limited information is available regarding factors acting on the peritrophic matrix (PM). This study aimed to investigate the role of a Bt chitin-binding protein (CBPA) that had been previously shown to be expressed at pH 9 in vitro and could therefore be expressed in the alkaline gut of lepidopteron larvae. A ∆cbpA mutant was generated that was 10-fold less virulent than wild-type Bt HD73 towards Ostrinia furnacalis neonate larvae, indicating its important role in infection. Purified recombinant Escherichia coli CBPA was shown to have a chitin affinity, thus indicating a possible interaction with the chitin-rich PM. A translational GFP-CBPA fusion elucidated the localization of CBPA on the bacterial surface, and the transcriptional activity of the promoter PcbpA was immediately induced and confirmed at pH 9. Next, in order to connect surface expression and possible in vivo gut activity, last instar Galleriamellonella (Gm) larvae (not susceptible to Bt HD-73) were used as a model to follow CBPA in gut expression, bacterial transit, and PM adhesion. CBPA-GFP was quickly expressed in the Gm gut lumen, and more Bt HD73 strain bacteria adhered to the PM than those of the ∆cbpA mutant strain. Therefore, CBPA may help to retain the bacteria, via the PM binding, close to the gut surface and thus takes part in the early steps of Bt gut interactions.

Transcription in the acetoin catabolic pathway is regulated by AcoR and CcpA in Bacillus thuringiensis.

Acetoin (3-hydroxy-2-butanone) is an important physiological metabolic product in many microorganisms. Acetoin breakdown is catalyzed by the acetoin dehydrogenase enzyme system (AoDH ES), which is encoded by acoABCL operon. In this study, we analyzed transcription and regulation of the aco operon in Bacillus thuringiensis (Bt). RT-PCR analysis revealed that acoABCL forms one transcriptional unit. The Sigma 54 controlled consensus sequence was located 12 bp from the acoA transcriptional start site (TSS). ß-galactosidase assay revealed that aco operon transcription is induced by acetoin, controlled by sigma 54, and positively regulated by AcoR. The HTH domain of AcoR recognized and specifically bound to a 13-bp inverted repeat region that participates in 30-bp fragment mapping 81 bp upstream of the acoA TSS. The GAF domain in AcoR represses enhancer transcriptional activity at the acoA promoter. Transcriptions of the aco operon and acoR were repressed by glucose via CcpA, and CcpA specifically bound to sequences within the acoR promoter fragment. In the acoABCL and acoR mutants, acetoin use was abolished, suggesting that the aco operon is essential for utilization of acetoin. The data presented here improve our understanding of the regulation of the aco gene cluster in bacteria.

Cry78Ba1, One Novel Crystal Protein from Bacillus thuringiensis with High Insecticidal Activity against Rice Planthopper.

The rice planthopper is a very important hemipteran pest that preys on rice and substantially affects the safety of rice production. Moreover, the long-term prevention and control of these pests with chemical pesticides has led to an increase in the resistance of the rice planthopper as well as serious environmental pollution and food safety problems. Bacillus thuringiensis (Bt) has been used for the efficient and green control of a variety of rice pests. Therefore, based on the high-throughput screening of Bt strains that are active against the rice planthopper, we found that Bt strain B4F11 showed certain insecticidal activity against Laodelphax striatellus Fallén, and we have identified a novel insecticidal protein Cry78Ba1 from the Bt strain B4F11, which is expected to provide the specific and safe control of the rice planthopper. The Cry78Ba1 protein is composed of 380 amino acid residues with a molecular weight of 42.55 kDa and contains conserved Ricin_B_Lectin and Toxin_10 superfamily domains. It displays high insecticidal activity against L. striatellus with a lethal concentration (LC50) of 9.723 µg/mL. More importantly, this Toxin_10-like protein does not display sequence homology to any known allergen and can be degraded and inactivated rapidly when heated at 90 °C and in simulated gastrointestinal fluid. In summary, Cry78Ba1 has great potential for applications in the efficient and safe prevention and control of the rice planthopper.

The stationary phase regulator CpcR activates cry gene expression in non-sporulating cells of Bacillus thuringiensis.

Cell differentiation within an isogenic population allows the specialisation of subpopulations and a division of labour. Bacillus thuringiensis is a spore-forming bacterium that produces insecticidal crystal proteins (Cry proteins) in sporulating cells. We recently reported that strain B. thuringiensis LM1212 presents the unique ability to differentiate into two subpopulations during the stationary phase: spore-formers and crystal-producers. Here, we characterised the transcriptional regulator CpcR responsible for this differentiation and the expression of the cry genes. cpcR is located on a plasmid that also harbours cry genes. The alignment of LM1212 cry gene promoters revealed the presence of a conserved DNA sequence upstream from the -35 region. This presumed CpcR box was also found in the promoter of cpcR and we showed that cpcR transcription is positively autoregulated. Electrophoretic mobility shift assays suggested that CpcR directly controls the transcription of its target genes by binding to the CpcR box. We showed that CpcR was able to direct the production of a crystal consisting of a heterologous insecticidal Cry protein in non-sporulating cells of a typical B. thuringiensis kurstaki strain. Moreover, the expression of cpcR induced a reduction in the sporulation of this B. thuringiensis strain, suggesting an interaction between CpcR and the sporulation regulatory networks.

Bacillus thuringiensis Vip1 Functions as a Receptor of Vip2 Toxin for Binary Insecticidal Activity against Holotrichia parallela.

Bacillus thuringiensis is a well-known entomopathogenic bacterium that produces vegetative insecticidal proteins (Vips, including Vip1, Vip2, Vip3, and Vip4) during the vegetative phase. Here, we purified Vip1 and Vip2 from B. thuringiensis and characterized the insecticidal effects of these protoxins. Bioassay results showed that a 1:1 mixture of Vip1Ad and Vip2Ag, purified by ion-affinity chromatography independently, exhibited insecticidal activity against Holotrichia parallela larvae, with a 50% lethal concentration value of 2.33 µg/g soil. The brush border membrane (BBM) in the midgut of H. parallela larvae was destroyed after feeding the Vip1Ad and Vip2Ag mixture. Vacuolization of the cytoplasm and slight destruction of BBM were detected with Vip2Ag alone, but not with Vip1Ad alone. Notably, Vip1Ad bound to BBM vesicles (BBMVs) strongly, whereas Vip2Ag showed weak binding; however, binding of Vip2Ag to BBMV was increased when Vip1Ad was added. Ligand blotting showed that Vip2Ag did not bind to Vip1Ad but bound to Vip1Ad-t (Vip1Ad was activated by trypsin), suggesting the activation of Vip1Ad was important for their binary toxicity. Thus, our findings suggested that Vip1Ad may facilitate the binding of Vip2Ag to BBMVs, providing a basis for studies of the insecticidal mechanisms of Vip1Ad and Vip2Ag.

Effect of the spoIIID mutation on mother cell lysis in Bacillus thuringiensis.

SpoIIID is a small, sequence-specific DNA-binding protein which can direct many genes' transcription and has an effect on spore formation in Bacillus subtilis. We investigated the role of SpoIIID in mother cell lysis in Bacillus thuringiensis. A ß-galactosidase assay based on the promoter fusions with lacZ indicated that the sigK gene was positively regulated by SpoIIID and σK negatively regulated the expression of sigE. The spoIIID mutant strain exhibited no mother cell lysis in Schaeffer's sporulation medium (SSM) but did in ½ Luria-Bertani (LB) medium. cwlC is an essential hydrolase gene for mother cell lysis. Moreover, the expression of a PcwlC-lacZ fusion in spoIIID mutant was proved to be higher in ½ LB medium than in SSM. HD (ΔspoIIID)(ΔcwlC) mutant was obtained by knocking out the cwlC gene in HD(ΔspoIIID) and displayed no mother cell lysis in both SSM and ½ LB mediums. The deletion of spoIIID decreased the crystal protein production in HD73. The expression of Porf1cry8E and P5014 promoter fusions with lacZ gene in the acrystalliferous HD-(ΔspoIIID) mutant showed similar activity to that in the acrystalliferous HD73- strain before T7 and slightly higher than that in the acrystalliferous HD73- after T7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Cry1Ac production in HD-(ΔspoIIID) directed by the Porf1cry8E and P5014 promoters was at a similar level as that in HD73 wild strain. Altogether, these results suggested that the spoIIID mutant with Porf1cry8E or P5014 promoters could be an alternative delivery system for cry gene expression with no mature spore formation and medium-dependent mother cell lysis.

Expression of cry genes in Bacillus thuringiensis biotechnology.

Bacillus thuringiensis is a gram-positive, spore-forming bacterium that produces insecticidal crystal proteins during sporulation. The production of these crystals results primarily from the expression of cry genes. In this review, we focus on the expression and application of cry genes directed by both cry gene promoters and non-cry gene promoters in different hosts. However, not all cry genes and niches are compatible with B. thuringiensis. New delivery systems offsetting the current limitations in B. thuringiensis application are needed to improve Cry production, niche fitness, and persistence. This review examines currently available research and highlights areas in need of further research and development for more effective production and utilization of Cry insecticidal proteins.

Protaetia brevitarsis larvae can efficiently convert herbaceous and ligneous plant residues to humic acids.

Utilization of the organic residues produced after crop harvesting is currently an important issue across the world. The edible insect Protaetia brevitarsis larvae can feed various organic matters. In this paper, we investigated the potential to utilize the insect to convert herbaceous and ligneous plant residues. We feed the insect larvae with maize straw and sawdust and analyzed the produced insect manure. P. brevitarsis larval was found to be able to digest both herbaceous and ligneous straw and insect manure extract shown no phytotoxicity. The mass fractions of humic acids (HAs) in the insect manure derived from maize straw and sawdust digestion were 24.37% and 14.46%, respectively. The 13C cross-polarization magic-angle spinning nuclear magnetic resonance (CP-MAS NMR) spectra data indicated that the HAs in the insect manure were similar to those found in the soil. These data suggested that P. brevitarsis larvae can be used to convert agricultural residues and produce organic fertilizers.

Cry78Aa, a novel Bacillus thuringiensis insecticidal protein with activity against Laodelphax striatellus and Nilaparvata lugens.

Transgenic plants expressing insecticidal proteins originating from Bacillus thuringiensis (Bt) have successfully been used to control lepidopteran and coleopteran pests with chewing mouthparts. However, only a handful of Bt proteins have been identified that have bioactivity against sap sucking pests (Hemiptera), including aphids, whiteflies, plant bugs and planthoppers. A novel Bt insecticidal protein with significant toxicity against a hemipteran insect pest is described here. The gene encoding the 359 amino acid, 40.7 kDa protein was cloned from strain C9F1. After expression and purification of the toxin, its median lethal concentration (LC50) values against Laodelphax striatellus and Nilaparvata lugens were determined as 6.89 µg/mL and 15.78 µg/mL respectively. Analysis of the toxin sequence revealed the presence of both Toxin_10 and Ricin_B_Lectin domains.