Thuringiensis HD-1 Research Articles

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.

Dose-response analysis of Bacillus thuringiensis HD-1 cry- spore reduction on surfaces using formaldehyde with pre-germination.

To establish a basis for rapid remediation of large areas contaminated with Bacillus anthracis spores. Representative surfaces of wood, steel and cement were coated by nebulization with B. thuringiensis HD-1 cry- (a simulant for B. anthracis) at 5.9± 0.2, 6.3± 0.2 and 5.8± 0.2 log10 CFU per cm2 , respectively. These were sprayed with formaldehyde, either with or without pre-germination. Low volume (equivalent to ≤2500 L ha-1 ) applications of formaldehyde at 30 g l-1 to steel or cement surfaces resulted in ≥4 or ≤2 log10 CFU per cm2 reductions respectively, after 2h exposure. Pre-germinating spores (500 mmol l-1 l-alanine and 25 mmol l-1 inosine, pH7) followed by formaldehyde application showed higher levels of spore inactivation than formaldehyde alone with gains of up to 3.4 log10 CFU per cm2 for a given dose. No loss in B. thuringiensis cry- viability was measured after the 2h germination period, however, a pre-heat shock log10 reduction was seen for B. anthracis strains: LSU149 (1.7 log10), Vollum and LSU465 (both 0.9 log10), LSU442 (0.2 log10), Sterne (0.8 log10) and Ames (0.6 log10). A methodology was developed to produce representative spore contamination of surfaces along with a laboratory-based technique to measure the efficacy of decontamination. Dose-response analysis was used to optimize decontamination. Pre-germinating spores was found to increase effectiveness of decontamination but requires careful consideration of total volume used (germinant and decontaminant) by surface type. To be practically achievable, decontamination of a wide area contaminated with B. anthracis spores must be effective, timely and minimize the amount of materials required. This study uses systematic dose-response methodology to demonstrate that such an approach is feasible.

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 β-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.

Creation of an Industrial Bacillus thuringiensis Strain With High Melanin Production and UV Tolerance by Gene Editing.

Bacillus thuringiensis produces insecticidal crystal proteins (ICPs) which exhibit strong insecticidal toxicity. But when used in the field, ICPs would be destroyed by ultraviolet (UV) radiation in sunlight, thus decreasing the insecticidal activity and shortening the persistence. To improve the duration of B. thuringiensis preparations, we endowed a highly toxic industrial B. thuringiensis HD-1 with UV tolerance by making it produce melanin, a pigment that absorbs UV radiation. In B. thuringiensis, melanin is derived from homogentisate (HGA), an intermediate in the tyrosine pathway. And the absence of homogentisate-1,2-dioxygenase (HmgA) will lead to the formation of melanin. In this study, we used the CRISPR/Cas9 system to knock out the hmgA gene and obtained a melanin-producing mutant HD-1-ΔhmgA from strain HD-1. The melanin yield by mutant HD-1-ΔhmgA reached 3.60 mg/mL. And the anti-UV test showed that melanin serves as a protection to both the organism and the ICPs. After UV irradiation for 3 h, mutant HD-1-ΔhmgA still had an 80% insecticidal activity against the cotton bollworm, Helicoverpa armigera, while the control line only had about 20%. This study creates a light-stable biopesticide prototype based on a classic industrial strain that can be applied directly and takes the melanin-producing strain as a concept to improve the preparation validity.

Combined transcriptomic and proteomic analysis of developmental features in the immune system of Plutella xylostella during larva-to-adult metamorphosis

Diamondback moth (DBM), Plutella xylostella L. (Lepidoptera: Plutellidae) is considered one of the most destructive worldwide agricultural pests and has developed various defence mechanisms to fight against the available pesticides. Understanding the host-defence system of P. xylostella is vital for developing biocontrol-based pest management strategies. Although there are several studies on P. xylostella, little is known about the changes in the immune system during the larva-to-adult metamorphosis. RNA-seq and iTRAQ investigations of P. xylostella from 2-day-old fourth instar larvae (L4D2), pupa (P0), and adult (A0) were done to understand these alterations at a molecular level. A total of 412/ 584 up-regulated and 1430/ 757 down-regulated genes/proteins between larva and pupa, 813/ 589 up-regulated and 1206/ 846 down-regulated genes/proteins between pupa and adult were identified. It was shown that the differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) expression were up-regulated during the pupation and emergence of metamorphosis. The pathway enrichment analysis demonstrated that DEGs and DEPs were mainly associated with the energy generation and metabolism and innate immunity of the insect. The expression of immune-related and developmental-related genes were significantly different during the developmental process of P. xylostella. Moreover, the expression of four focused genes, i.e., serine proteinase inhibitor (Serpin-15), prophenoloxidase activating proteinase 1 (PAP-1) and 3a (PAP-3a), Gram-negative bacteria-binding protein (GNBP-6), was different in developmental stages and after Bacillus thuringiensis HD73 and Metarhizium anisopliae infection. The phenoloxidase (PO) activity in plasma was also significantly up-regulated during the pathogen infection. Recombinant proteins PAP-1, PAP-3a, GNBP-6 could significantly trigger the PO activity in vitro, Serpin-15 could suppress the PO activity. Taken together, these results indicate that Serpin-15, PAP-1, PAP-3a, and GNBP-6 might have the potential for co-regulation of immunity and development in P. xylostella. In conclusion, this study provided the immune system dynamics in the developmental process of P. xylostella and identified four candidate genes that can serve as potential targets for pest control strategies.

Expression of thurincin H, ChiA74 and Cry proteins at the sporulation phase in Bacillus thuringiensis HD1.

The objective of this study was to produce thurincin H, ChiA74 and Cry proteins together using Bacillus thuringiensis subsp. kurstaki HD1 as a heterologous host. pSTAB-ThurH and pSTAB-ChiA74 constructs were designed to produce thurincin H and chitinase, respectively, at the sporulation phase. They were transformed into Bt HD1 generating the recombinant strains HD1/pSTAB-ThurH and HD1/pSTAB-ThurH/pSTAB-ChiA74. Antimicrobial and chitinolytic activity tests were performed with recombinant strains. Both strains were able to produce thurincin H up to 72h with antibacterial activity of ~4000Umg-1 . The HD1/pSTAB-ThurH/pSTAB-ChiA74 strain also showed chitinolytic activity of ~23mUmg-1 at 72h. All B. thuringiensis strains exhibited crystal formation at 72, and 96h. In addition, the application of thurincin H in corn seeds increased the germination percentage and root length by 7% and 10%, respectively. We showed that is possible to produce three proteins of biotechnological interest at the sporulation stage in B. thuringiensis, which two of them (thurincin H, and ChiA74) are naturally expressed in the vegetative stage. These results form the basis for developing of a biocontrol and biostimulator product that can be used as an alternative for chemical application.

Expression of ChiA74∆sp and its truncated versions in Bacillus thuringiensis HD1 using a vegetative promoter maintains the integrity and toxicity of native Cry1A toxins

Our objective was to determine whether a recombinant chitinase ChiA74∆sp of Bacillus thuringiensis and its truncated versions (ChiA74∆sp-60, ChiA74∆sp-50) could be produced in B. thuringiensis HD1 with no detrimental effect on the size and insecticidal activity of the native bipyramidal Cry crystal. chiA-p, the promoter used to drive chitinase gene expression, was active during vegetative growth of Cry-B. HD1 recombinants showed increases from ~7- to 12-fold in chitinase activity when compared with parental HD1 and negligible or no effect on the volume of bipyramidal crystals was observed. HD1/ChiA74∆sp-60 showed increases from 20% to 40% in the yield of Cry1A per unit of culture medium when compared with parental HD1 and HD1/ChiA74∆sp-50, HD1/ChiA74∆sp. Inclusion bodies presumably composed of the enzyme attached to native Cry1A crystals of recombinant strains were observed; these inclusions were likely responsible for the enhancements in chitinase activity. Western blot analysis using polyclonal anti-ChiA74∆sp showed a weak signal with proteins of ~50 kDa in sporulated and lysed cells of recombinant strains. Bioassays against Spodoptera frugiperda using sporulated/lysed samples of the recombinant strains did not show statistically significant differences in LC50s when compared with HD1.

Genomic Analysis of the Recent Viral Isolate vB_BthP-Goe4 Reveals Increased Diversity of φ29-Like Phages.

We present the recently isolated virus vB_BthP-Goe4 infecting Bacillus thuringiensis HD1. Morphological investigation via transmission electron microscopy revealed key characteristics of the genus Phi29virus, but with an elongated head resulting in larger virion particles of approximately 50 nm width and 120 nm height. Genome sequencing and analysis resulted in a linear phage chromosome of approximately 26 kb, harbouring 40 protein-encoding genes and a packaging RNA. Sequence comparison confirmed the relation to the Phi29virus genus and genomes of other related strains. A global average nucleotide identity analysis of all identified φ29-like viruses revealed the formation of several new groups previously not observed. The largest group includes Goe4 and may significantly expand the genus Phi29virus (Salasvirus) or the Picovirinae subfamily.

A strong promoter of a non-cry gene directs expression of the cry1Ac gene in Bacillus thuringiensis.

Bacillus thuringiensis bacteria show insecticidal activities that rely upon the production of insecticidal crystal proteins, which are encoded by cry or cyt genes and can target a variety of insect pests. It has been shown that cry1Ac is the only cry gene in B. thuringiensis subsp. kurstaki HD73 (B. thuringiensis HD73) and its expression is controlled by both σE and σK. Here, we report a novel σE-dependent strong promoter of a non-cry gene (HD73_5014), which can direct strong cry1Ac gene expression in B. thuringiensis HD73. We constructed an E. coli-B. thuringiensis shuttle vector (pHT315-P 5014 -1Ac) for cry1Ac gene expression, using the HD73_5014 gene promoter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis showed that expression of the cry1Ac gene directed by the HD73_5014 gene promoter was at the same level as that directed by the previously known strongest cry promoter, P cry8E . However, this strain did not form typical bipyramidal crystals in mother cells, as observed by transmission electron microscopy and atomic force microscope. The strain with Cry1Ac protein expression under the control of the HD73_5014 gene promoter (P 5014 -cry1Ac) showed insecticidal activity against Plutella xylostella similar to that under the control of the orf1cry8E gene promoter (P cry8E -cry1Ac). Collectively, these results suggest that the HD73_5014 gene promoter, as a non-cry gene promoter, would be an efficient transcriptional element for cry gene expression. These data also show the possibility for improving Cry production by searching for transcriptional elements in not only cry genes, but also non-cry genes.

Nanopore based detection of Bacillus thuringiensis HD-73 spores using aptamers and versatile DNA hairpins.

A versatile nanopore sensing platform to detect any aptamer using nanopores by designing DNA hairpins that are complementary to the aptamer is described. This platform can detect the presence of aptamer binding targets regardless of their size, which has been a major hurdle for nanopore detection systems. Moreover, the signal-to-noise ratio is increased by eliminating most of the unwanted substances from the sample via simple sample preparation steps. To detect Bacillus thuringiensis HD-73 spores using this sensing platform, DNA hairpins complementary to the target-specific aptamers were designed, and the hairpins were characterized using alpha-hemolysin nanopores after the reaction of spores and aptamers and subsequent reaction with the complementary DNA hairpins. The platform exhibited a detection limit as low as 1.2 × 101 CFU mL-1 and was compatible with a wide range of spore concentrations from 1.2 × 101 CFU mL-1 to 1.2 × 106 CFU mL-1 while it is still expandable to higher spore concentrations.

Selection and characterization of Bacillus thuringiensis strains from northwestern Himalayas toxic against Helicoverpa armigera.

In this study, we present the selection and the characterization of Bacillus thuringiensis (Bt) strains with respect to their cry/cyt gene content and toxicity evaluation toward one of the most important polyphagous lepidopteran pest, Helicoverpa armigera. Fifty‐six Bt isolates were obtained from 10 different regions of northwestern Himalayas, recording a total B. thuringiensis index of 0.62. Scanning electron microscopy revealed presence of bipyramidal, spherical, flat and irregular crystal shapes; SDS‐PAGE analysis of spore‐crystal mixtures showed the prominence of 130, 70, and 100 kDa protein bands in majority of the isolates; PCR analysis with primers for eight cry and cyt gene families and 13 cry gene subfamilies resulted in isolates showing different combinations of insecticidal genes. Strains containing cry1 were the most abundant (57.1%) followed by cyt2 (46.42%), cry11 (37.5%), cry2 (28.57%), cry4 (21.42%), cyt1 (19.64%), cry3 (8.9%), and cry7, 8 (7.14%). A total of 30.35% of the strains did not amplify with any of the primers used in this study. Median lethal concentration 50 (LC50) estimates of spore‐crystal mixtures of Bt‐JK12, 17, 22, 48, and 72 against second instar larvae of H. armigera was observed to be 184.62, 275.39, 256.29, 259.93 μg ml−1, respectively. B. thuringiensis presents great diversity with respect to the presence of crystal protein encoding genes and insecticidal activity. Four putative toxic isolates identified in this study have potential application in insect pest control. B. thuringiensis isolate JK12 exhibited higher toxicity against H. armigera than that of B. thuringiensis HD1, hence can be commercially exploited to control insect pest for sustainable crop production. The results of this study confirm the significance of continuous exploration of new Bt stains from different ecological regions of the world.

Translocation of Bacillus thuringiensis in Phaseolus vulgaris tissues and vertical transmission in Arabidopsis thaliana.

To demonstrate the ability of Bacillus thuringiensis to penetrate as spore-crystal complex to the internal tissues of bean plants, and keep its insecticidal activity. To test the vertical transmission of the spore-crystal complex in Arabidopsis thaliana. The experimental strain was transformed with the pMUTIN-gfp plasmid which labelled the spores of B. thuringiensis HD-73 with the GFP protein. Once the rhizosphere of the bean plants was inoculated with the labelled strain, the bacterium was recovered from leaves, stems, and petioles. Furthermore, toxicity of treated plants was significantly higher than control plants when bio-assayed on cabbage looper larvae. The labelled strain was recovered from the dead insects. When the rhizosphere of A. thaliana plants was inoculated with the labelled strain, mature seeds from these plants were surface-sterilized and grown under invitro conditions. The labelled strain was recovered from the seedlings. We showed that B. thuringiensis subsp. kurstaki (HD-73) in the rhizosphere can translocate to upper tissues of bean plants, and keep its insecticidal activity. Transmission of the labelled B. thuringiensis strain passed to the next generation of A. thaliana. The role of B. thuringiensis as a potential facultative endophyte bacterium and the possible biotechnological repercussions are discussed.

Cloning and characterisation of a novel cry1H gene effective against lepidopterous larvae from a Bacillus thuringiensis strain

We characterised a novel holotype cry gene (cry1Hc1) harboured in BN23-5 Bt strain which was isolated from soil samples in Sichuan, China. In this study, the full length of the cry1Hc1 gene was cloned from the strain. The cry1Hc1 gene was inserted into a shuttle vector (pSTK) and expressed in an acrystalliferous mutant Bacillus thuringiensis HD73−. In this transformant, cry1Hc1 was expressed and diamond-shaped parasporal crystals were formed. The resulting insecticidal activity showed that the Cry1Hc1 protein exhibited high larvicidal activity against larvae of Plutella xyllostella and Ostrinia furnacalis with lethal concentration 50 of 55.21 and 118.44 μg/ml, respectively. Sequence analysis of the gene was also performed; the Cry1Hc1 protein retained eight conserved regions commonly found in the existing Cry proteins.

Three-dimensional (3D) structure prediction and function analysis of the chitin-binding domain 3 protein HD73_3189 from Bacillus thuringiensis HD73.

Bacillus thuringiensis (Bt) is capable of producing a chitin-binding protein believed to be functionally important to bacteria during the stationary phase of its growth cycle. In this paper, the chitin-binding domain 3 protein HD73_3189 from B. thuringiensis has been analyzed by computer technology. Primary and secondary structural analyses demonstrated that HD73_3189 is negatively charged and contains several α-helices, aperiodical coils and β-strands. Domain and motif analyses revealed that HD73_3189 contains a signal peptide, an N-terminal chitin binding 3 domains, two copies of a fibronectin-like domain 3 and a C-terminal carbohydrate binding domain classified as CBM_5_12. Moreover, analysis predicted the protein's associated localization site to be the cell wall. Ligand site prediction determined that amino acid residues GLU-312, TRP-334, ILE-341 and VAL-382 exposed on the surface of the target protein exhibit polar interactions with the substrate.

The specificity of immune priming in silkworm, Bombyx mori, is mediated by the phagocytic ability of granular cells

In the past decade, the phenomenon of immune priming was documented in many invertebrates in a large number of studies; however, in most of these studies, behavioral evidence was used to identify the immune priming. The underlying mechanism and the degree of specificity of the priming response remain unclear. We studied the mechanism of immune priming in the larvae of the silkworm, Bombyx mori, and analyzed the specificity of the priming response using two closely related Gram-negative pathogenic bacteria (Photorhabdus luminescens TT01 and P. luminescens H06) and one Gram-positive pathogenic bacterium (Bacillus thuringiensis HD-1). Primed with heat-killed bacteria, the B. mori larvae were more likely to survive subsequent homologous exposure (the identical bacteria used in the priming and in the subsequent challenge) than heterologous (different bacteria used in the priming and subsequent exposure) exposure to live bacteria. This result indicated that the B. mori larvae possessed a strong immune priming response and revealed a degree of specificity to TT01, H06 and HD-1 bacteria. The degree of enhanced immune protection was positively correlated with the level of phagocytic ability of the granular cells and the antibacterial activity of the cell-free hemolymph. Moreover, the granular cells of the immune-primed larvae increased the phagocytosis of a previously encountered bacterial strain compared with other bacteria. Thus, the enhanced immune protection of the B. mori larvae after priming was mediated by the phagocytic ability of the granular cells and the antibacterial activity of the hemolymph; the specificity of the priming response was primarily attributed to the phagocytosis of bacteria by the granular cells.

Identification of metabolism pathways directly regulated by sigma(54) factor in Bacillus thuringiensis.

Sigma54 (σ54) regulates nitrogen and carbon utilization in bacteria. Promoters that are σ54-dependent are highly conserved and contain short sequences located at the −24 and −12 positions upstream of the transcription initiation site. σ54 requires regulatory proteins known as bacterial enhancer-binding proteins (bEBPs) to activate gene transcription. We show that σ54 regulates the capacity to grow on various nitrogen sources using a Bacillus thuringiensis HD73 mutant lacking the sigL gene encoding σ54 (ΔsigL). A 2-fold-change cutoff and a false discovery rate cutoff of P < 0.05 were used to analyze the DNA microarray data, which revealed 255 genes that were downregulated and 121 that were upregulated in the ΔsigL mutant relative to the wild-type HD73 strain. The σ54 regulon (stationary phase) was characterized by DNA microarray, bioinformatics, and functional assay; 16 operons containing 47 genes were identified whose promoter regions contain the conserved −12/−24 element and whose transcriptional activities were abolished or reduced in the ΔsigL mutant. Eight σ54-dependent transcriptional bEBPs were found in the Bt HD73 genome, and they regulated nine σ54-dependent promoters. The metabolic pathways activated by σ54 in this process have yet to be identified in Bacillus thuringiensis; nonetheless, the present analysis of the σ54 regulon provides a better understanding of the physiological roles of σ factors in bacteria.

Bacillus thuringiensis HD-1 Cry- : development of a safe, non-insecticidal simulant for Bacillus anthracis.

A representative simulant for spores of Bacillus anthracis is needed for field testing. Bacillus thuringiensis is gaining recognition as a suitable organism. A strain that does not form the insecticidal, parasporal crystals that are characteristic of this species is a more accurate physical representative of B. anthracis spores. We developed noninsecticidal derivatives of two isolates of B. thuringiensis HD-1. Two plasmid-cured derivatives of B. thuringiensis HD-1, unable to make crystal toxins ('Cry(-) '), were isolated. These isolates and the existing Cry(-) strain, B. thuringiensis Al Hakam, were probed with PCR assays against the known insecticidal genes cry, vip and cyt. Their genomic DNA was sequenced to demonstrate a lack of insecticidal genes. This was confirmed by bioassays against a number of invertebrate species. Real-time PCR assays were developed to identify the B. thuringiensis HD-1 Cry(-) derivatives and an effective differential and selective medium was assessed. All three Cry(-) isolates are devoid of known insecticidal determinants. The B. thuringiensis HD-1 Cry(-) derivatives can easily be recovered from soil and identified by PCR with some selectivity. The B. thuringiensis HD-1 Cry(-) derivatives represent accurate, nongenetically manipulated simulants for B. anthracis with excellent human and environmental safety records.

Priming Galleria mellonella (Lepidoptera: Pyralidae) larvae with heat-killed bacterial cells induced an enhanced immune protection against Photorhabdus luminescens TT01 and the role of innate immunity in the process.

The current study investigated the characteristics and mechanism of the invertebrate immune priming using Galleria mellonella (L.) (Lepidoptera: Pyralidae) larvae (host) and Photorhabdus luminescens TT01 (pathogen) as a model. The following parameters of the G. mellonella larvae primed by hemocoel injection of heat-killed cells of TT01 or Bacillus thuringiensis HD-1 were determined at designated times after priming and then compared and analyzed systematically: mortality of the primed larvae against TT01 infection (immune protection level), hemocyte density, phagocytosis and encapsulation abilities ofhemocyte, and antibacterial activity of cell free hemolymph (major innate parameters). The results showed that 1) immune priming increased survival of the larvae against a lethal infection of TT01 and the levels and periods of protection correlated positively to the priming dose; 2) the changes on the levels of protection and the major innate parameters of the larvae primed with either TT01 or HD-1 followed a similar pattern of the convex curve, although the levels and the timing of changes differed significantly among the four innate immune parameters and between two priming bacteria; and 3) the immune protection level at a time after priming was correlated to the overall level of four innate immune parameters of the primed larvae. The current study demonstrated that the immune priming phenomenon of G. mellonella larvae has low level of specificity, and it was achieved mainly by the regulation on the quantity and activity of major innate immune parameters, such as hemocytes, antimicrobial peptides, and enzymes.

Bacillus thuringiensis subsp. sichuansis strain MC28 produces a novel crystal protein with activity against Culex quinquefasciatus larvae

The Bacillus thuringiensis subsp. sichuansis MC28 strain produces spherical parasporal crystals during sporulation and exhibits remarkable insecticidal activity against dipteran and lepidopteran pests. We characterized a novel cry gene (cry69Aa1), which was found in the pMC95 plasmid of the MC28 strain. The cry69Aa1 gene was inserted into a shuttle vector (pSTK) and expressed in an acrystalliferous mutant B. thuringiensis HD73⁻. In this transformant, a large number of spherical parasporal crystals, which were toxic to Culex quinquefasciatus (Diptera), were formed.

Hyperproduction of chitinase influences crystal toxin synthesis and sporulation of Bacillus thuringiensis

Bacillus thuringiensis HD-73 was transformed with the endochitinase gene chiA74 under the control of a strong promoter (pcytA) and a 5' mRNA stabilizing (STAB-SD) sequence (HD-73-pEBchiA74). Expression levels were compared with those observed from the wild type strain (HD-73) and the recombinant HD-73 strain expressing chiA74 under the control of its native promoter (HD-73-pEHchiA74). The chitinolytic activity of HD-73-pEBchiA74 was markedly elevated, being ~58- and 362-fold higher than, respectively, HD-73-pEHchiA74 and parental HD-73, representing the highest levels of chitinase expression in recombinant B. thuringiensis reported to date. Parasporal crystals measured under transmission electron microscopy showed that HD-73 produced crystals of 1.235 (+/-0.214) and 1.356 (+/-0.247) mum in length when the bacterium was grown in respectively, NBS and NBS with glucose. Otherwise, HD-73-pEBchiA74 synthesized crystals of 1.250 (+/-0.222) and 1.139 (+/-0.202) mum in length when cultivated in NBS and NBS with glucose, respectively, values that showed a diminution of ~10 and 20% compared with crystals produced by HD-73-pEHchiA74 grown under the same conditions. Comparison of viable spore counts per ml showed that HD-73-pEBchiA74 produced fewest viable spores (1.5 x 10(9), 1.3 x 10(9)), compared to HD-73-pEHchiA74 (4.9 x 10(9), 5.3 x 10(9)) and HD-73 (6.8 x 10(9), 8.8 x 10(9)) when grown in NBS and NBS supplemented with glucose, respectively. No change in cellular protease activity was observed despite the overproduction of the chitinase.