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

Accepted Manuscript 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

Karen S. González-Ponce, L.E. Casados-Vázquez, Paulina Lozano-Sotomayor, Dennis K. Bideshi, Ma. Cristina del RincónCastro, José E. Barboza-Corona PII: DOI: Reference:

S0141-8130(18)35766-0 https://doi.org/10.1016/j.ijbiomac.2018.11.173 BIOMAC 11050

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

24 October 2018 16 November 2018 17 November 2018

Please cite this article as: Karen S. González-Ponce, L.E. Casados-Vázquez, Paulina Lozano-Sotomayor, Dennis K. Bideshi, Ma. Cristina del Rincón-Castro, José E. BarbozaCorona , 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. Biomac (2018), https://doi.org/10.1016/j.ijbiomac.2018.11.173

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ACCEPTED MANUSCRIPT

Submitted to International Journal of Biological Macromolecules

Corresponding Author: Dr. José E. Barboza-Corona Posgrado en Biociencias Departamento de Alimentos División de Ciencias de la Vida Universidad de Guanajuato Irapuato, Guanajuato, MEX. 36500 Tel: +52-462-624-1889 Email: [email protected]

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Revised version November 16, 2018

Expression of ChiA74∆sp and its truncated versions in Bacillus

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thuringiensis HD1 using a vegetative promoter maintains the

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integrity and toxicity of native Cry1A toxins Karen S. González-Ponce1, L.E. Casados-Vázquez1,2, Paulina Lozano-Sotomayor3, Dennis

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K. Bideshi4,5, Ma. Cristina del Rincón-Castro1,2, José E. Barboza-Corona1,2*

Graduate Program in Biosciences, Life Science Division, University of Guanajuato

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Campus Irapuato-Salamanca. Irapuato, Guanajuato, 36500, México. Food Department, Life Science División, University of Guanajuato Campus Irapuato-

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Salamanca. Irapuato, Guanajuato, 36500, México. Department of Chemistry, Division of Natural and Exact Sciences, University of

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Guanajuato Campus Guanajuato, 36250, México. Department of Biological Sciences, California Baptist University, 8432 Magnolia

Avenue, Riverside, CA, 92504, USA. 5

Department of Entomology, University of California, Riverside, CA 92521, USA.

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ACCEPTED MANUSCRIPT ABSTRACT 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

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the native bipyramidal Cry crystal. chiA-p, the promoter used to drive chitinase gene

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expression, was active during vegetative growth of Cry-B. HD1 recombinants showed

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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.

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HD1/ChiA74∆sp-60 showed increases from 20% to 40% in the yield of Cry1A per unit of

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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

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Cry1A crystals of recombinant strains were observed; these inclusions were likely

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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

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and lysed cells of recombinant strains. Bioassays against Spodoptera frugiperda using

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sporulated/lysed samples of the recombinant strains did not show statistically significant

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differences in LC50s when compared with HD1.

Keywords: Bacillus thuringiensis; chitinase ChiA74; Cry proteins; truncated versions

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ACCEPTED MANUSCRIPT 1. Introduction Bacillus thuringiensis is a sporogenic bacterium that synthesizes a plethora of insecticidal proteins (Cry, crystalline; Cyt, cytolytic) which interact through disulfide bonds or electrostatic interactions to produce crystalline inclusions, also called parasporal bodies,

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observable by phase contrast microscopy. Parasporal bodies have different morphologies

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and sizes, but among the most common are those with a bipyramidal shape, including those

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that associate with small Cry2Aa cuboidal inclusions of B. thuringiensis kurstaki HD1 1.

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In addition to Cry and Cyt proteins, chitinolytic enzymes of B. thuringiensis have gained interest in industry as these enzymes can be used to generate useful chitin-derived

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oligosaccharides, synergize insecticidal activities of Cry proteins, and for their inhibitory effect against phytopathogenic fungi 2,3. Low yields of chitinases occur in B.

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thuringiensis, even when culture media are supplemented with chitin. However,

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enhancements in synthesis of these enzymes have been achieved through genetic

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manipulations of their corresponding genes 4-8. In nature, bacterial chitinases are secreted by a process mediate by a N-terminal

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secretion signal peptide. Deletion of the signal peptide of recombinant chitinases results in

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intracellular accumulation of these enzymes in amorphous inclusion bodies (IBs) 9,10. In B. thuringiensis, these IBs are released together with Cry crystals and spores following autolysis 11-13. Among chitinases of B. thuringiensis is ChiA74, an enzyme synthesized by the native Mexican strain of B. thuringiensis subspecies kenyae (LBIT147). Immature ChiA74 has a mass of 74 kDa, but after translocation and secretion its signal peptide is cleaved to generate a mature enzyme of ~70 kDa which retains its catalytic (CD), chitinase insertion (CID), fibronectin-like (FnIII) and chitin binding (CBD) domains 14,15. A

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ACCEPTED MANUSCRIPT recent report on two truncated versions of ChiA74 lacking the CBD (ChiA74Δsp-60) or CBD /FnIII domains (ChiA74Δsp-50) synthesized in Escherichia coli, demonstrated that ChiA74Δsp-50 had similar activity as mature ChiA74Δsp when colloidal chitin was used as substrate. It was also shown that ChiA74∆sp and its truncated versions had chitinolytic

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activity against synthetic and natural substrates 15. Moreover, expression of chiA74∆sp

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using the strong cytA-p/STAB-SD promoter increased yield of the enzyme but reduced the

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size of the native Cry crystalline inclusion by ~30% in recombinant HD1, relative to the

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parental strain 12. In this regard, whether increases in ChiA74 without a corresponding decrease in Cry crystal synthesis could be achieved is unknown. Therefore, the objective of

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the present study was to determine whether ChiA74∆sp and its artificial truncated versions (ChiA74∆sp-60, ChiA74∆sp-50) could be produced in biologically active forms

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synchronously with native Cry crystals in HD1, and whether recombinant HD1 has

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improved toxicity against Spodoptera frugiperda, a common lepidopteran pest in

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agriculture.

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2. Materials and methods 2.1. Bacterial strains

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Escherichia coli TOP10 (Invitrogen, Carlsbad CA, USA) and E. coli ET12567 16 were used to generate chitinase truncated gene versions and to obtain non-methylated DNA, respectively. B. thuringiensis subsp. kurstaki HD1 (hereafter HD1) and B. thuringiensis Cry-B (hereafter Cry-B), were transformed with the chiA74∆sp artificial truncated versions. Cry-B is a plasmid-cured strain derived from HD-1. Recombinant E. coli strains were cultivated at 37ºC in Luria-Bertani (LB) broth (Invitrogen, Carlsbad CA, USA)

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ACCEPTED MANUSCRIPT supplemented with ampicillin (100 µg/mL), whereas B. thuringiensis were grown at 28ºC in Nutrient broth (NB) supplemented with erythromycin (25 µg/mL). To obtain growth curves of HD1 and Cry-B, one colony of each bacterium was used to inoculate NB at 28ºC, 200 rpm, to achieve an optical density of 0.5 at 600 nm. An aliquot of each synchronous

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culture was used to inoculate fresh NB, which was then incubated at 28ºC, 200 rpm.

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Aliquots were collected in triplicate at different times for monitoring growth at 600 nm.

2.2 Construction of chimeric chiA74ΔspΔtt-gfp under the control of different promoters of

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B. thuringiensis

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Amplification of chiA-p, cytA-p/STAB-SD and BtI/BtII-p was performed by PCR using chiA-1/chiA-2, Bti-II-1/BtI-II-2 and Cty-stab-1/Cyt-stab-2 primers, respectively (Table 1).

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Promoters chiA-p and BtI/BtII-p were amplified from genomic DNA of B. thuringiensis

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subsp kenyae (LBIT-82) 14 and HD1, respectively, whereas cytA-p/STAB-SD was obtained from pEBchiA74∆ps 12. The integrity of each promoter was confirmed by

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sequencing. Construct chiA74ΔspΔtt-gfp is a chimeric fusion composed of the open reading

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frame (ORF) of chitinase chiA74 lacking the signal peptide, stop codon, and transcriptional terminator, and the ORF of the green fluorescent protein gene (gfp) that was amplified

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using substrate pEBchiA74Δsp-gfp 12 and primers ChiA74-1 and GFP-RW. PCR amplifications were performed with the Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Waltham Massachusetts, USA). Promoters were cut with EcoRI and PstI and ligated into the pHT3101 digested with the same enzymes. pHT3101-promoters and chiA74ΔspΔtt-gfp were cut with PstI and ligated with T4 DNA ligase (New England BioLabs, Beverly, MA), and then used to transformed E. coli Top 10 (Invitrogen, Carlsbad

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ACCEPTED MANUSCRIPT CA, USA) to obtain the three recombinant plasmids with variant chiA74 under the control of different promoters (Fig. 1). The fidelity of the constructs and the correct orientation of chiA74ΔspΔtt-gfp was confirmed by restriction enzymes analysis, PCR and sequencing.

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2.3. Transformation of B. thuringiensis

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Plasmids were purified using the Fast-n-Easy Plasmid Mini-Prep Kit (Jena

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Bioscience, Jena, Alemania). Approximately 300 ng were mixed with 300 L of competent B. thuringiensis suspension kept on ice, followed by electroporation using a BTX ECM630

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Electro Cell Manipulator (San Diego, CA, USA) set at 2.3 kV, 475  and 25 F 12. After

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the pulse, 1 mL of Brain Heart Infusion (BHI) broth was added (Bioxon México) and the culture was incubated with gentle shaking for 2 h at 37°C. Transformants were selected on

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Nutrient agar (NA) supplemented with erythromycin (25 g/ml).

2.4 Comparison of promoter expression in B. thuringiensis

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Recombinant Cry-B harboring the chimeric chiA74ΔspΔtt-gfp under the control of

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different promoters (Fig. 1) was cultivated in NB supplemented with erythromycin at 28ºC

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and 200 rpm for 96 h. Bacteria in aliquots of 5 mL or 1.5 mL harvested at different times were centrifuged and washed 3x with double distilled water. Pellets were resuspended in 500 L of 100 mM Tris-HCl, pH 8, 150 mM NaCl or 100 mM phosphate buffer, pH 7, to assay GFP fluorescence or chitinase activity, respectively. Cells were sonicated (4x, 30 s each) at an amplitude of 30 Hz in a 20 kHz ultrasonic processor (Sonic and Materials, Inc, USA). Samples were centrifuge for 30 min at 5000 g and the protein concentration was determined in supernatants with the Quick Start Bradford kit (Bio-Rad, Hercules, CA,

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ACCEPTED MANUSCRIPT USA). Protein concentration were normalized in all samples, and the relative fluorescence (RFU) emitted by the GFP was recorded in a Synergy HTX Biotek (Winooski, VT, USA) using the following reaction mix: 5 L (0.014 mg) sample, 145 L 100 mM Tris-HCl, 150 mM NaCl, and the fluorogenic substrate 4-MU-(GlcNAc)3 (Sigma, St. Louis, MO) at pH

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7.0, as previously described 15.

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2.5. Construction of truncated chitinase versions

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The recombinant plasmid pHT3101-chiAp-chiA74Δsp 12 was used as the template to generate these constructs. The plasmid harbors the endochitinase chiA74 regulated by its

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native promoter (chiA-p), its ribosome binding site (RBS) and transcriptional terminator, but the ORF lacked the signal peptide sequence necessary for translocation of the chitinase

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in B. thuringiensis 12. To generate truncated versions of chiA74Δsp under the control of

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chiA-p, promoter chiA-p was amplified from pHT3101-chiAp-chiA74Δsp using the primers

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pchiA1 and pchiA2 (Table 1). The amplicon was digested with EcoRI and PstI and ligated in pHT3101 to obtain pHT3101-chiA-p. Primer pairs (a) N-termchiA, CAT/CID and (b) N-

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termchiA, CAT/FnIII, (Table S1), were used to amplify chiA74Δsp lacking the chitinbinding domain (chiA74ΔspΔCBD, chiA74Δsp-60) and the fibronectin-like domain

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(chiA74ΔspΔCBDΔFnIII, chiA74Δsp-50), respectively 15 (Fig. 2). PCR amplification was performed with Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Waltham Massachusetts, USA) in a C1000 Touch Thermal Cycler (Bio-Rad, Hercules, CA, USA) using the following conditions: 98°C for 30 s, followed by 30 cycles of 10 s at 98°C, 30 s at 55°C, 45 s for chiA74Δsp-50 and 1 min for chiA74Δsp-60 at 72°C, with a final extension cycle at 72°C for 5 min. Amplicons were purified using the PCR purification kit (Jena

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ACCEPTED MANUSCRIPT Bioscience, Jena, Germany), digested with PstI and then ligated into the pHT3101-chiA-p cut with the same enzyme using T4 DNA ligase (New England BioLabs, Beverly, MA) to generate pHT3101-chiA-p-chiA74Δsp-60 and pHT3101-chiAp-chiA74Δsp-50 (Fig. 2). Subsequently,

the

transcriptional

terminator

of

chiA74

was

amplified

using

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oligonucleotides termchi1 and termchi2 (Table 1), as follows: 98°C for 30 s, followed by

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30 cycles (10 s at 98°C, 30 s at 55°C, 30s at 72°C), with a final extension of 5 min at 72°C.

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Amplicons and plasmids were digested with SphI, and ligated; all constructs had the

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transcriptional terminator of chiA74.

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2.6. Chitinase activity of recombinant strains of B. thuringiensis Recombinant strains and controls (Cry-B and HD1) were cultivated in NB with or

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without erythromycin (25 μg/mL) at 28°C, 200 rpm until >95% of cells sporulated and

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lyse. One milliliter of each culture was collected, centrifuged at 13000g for 20 min and the pellet washed 3x with double distilled water. Pellets (spores, Cry crystals and putative

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chitinase inclusions) were solubilized in 150 L of solubilization buffer [30 mM Na2CO3,

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0.2% (p /v) β-ME, pH 9], as described previously Barboza-Corona et al. (2014) 12. Suspensions were incubated at 37°C with gentle agitation at 180 rpm for 40 min,

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centrifuged, and protein concentration was determined using the Quick Start kit (Bradford, Bio-Rad, Hercules, CA, USA). Chitinase activity was evaluated with two different substrates. The first method employed the fluorogenic chitin derivate 4-MU-(GlcNAc)3 (Sigma, St. Louis, MO), as described previously Juárez-Hernández et al. (2017) 15. The amount of 4-MU released from the substrate was calculated spectrofluorometrically (excitation at 360 nm and emission at 455 nm) with a Synergy HTX Biotek (Winooski, VT)

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ACCEPTED MANUSCRIPT instrument using a 4-MU standard curve; one unit (U) of chitinase activity was defined as the amount the enzyme required to release 1 mol of 4-methylumbelliferone in 1 h. The second method employed 0.5% colloidal chitin (Sigma-Aldrich, St. Louis MO, USA) which was mixed with 0.06 mg protein/mL to reach a final volume of 0.5 mL with 20 mM

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phosphate buffer (pH 7), followed by incubation at 37 °C with gentle agitation. Aliquots of

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40 μL were taken after 24 h of incubation and mixed with the same volume of 3,5-

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dinitrosalicylic acid. Samples were boiled for 5 min, cooled and double distilled water was

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added to achieve a final volume of 200 μL. Finally, absorbance was measured at 540 nm in a Synergy HTX Biotek (Winooski, VT, EE.UU.) to determine the concentration of reducing

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sugars. One Unit of activity was defined as the amount of enzyme required to release 1

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mol of N-acetylglucosamine (NAG) in 1 h.

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2.7. Protein and Western blot analysis

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After autolysis, 1.5 mL of NB cultures was harvested and spores, crystals, chitinase inclusion and cellular debris were pelleted by sedimentation at 13,000g for 20 min. Pellets

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were washed with double-distilled water, resuspended in 150 L of 5x Laemmli sample

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buffer and boiled for 5 min. Aliquots of 7 L were fractionated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) in a 10% gel [17]. Protein mass was estimated by comparison with the PageRuler unstained protein ladder (ThermoFisher Scientific, Waltham, Massachusetts, USA) and Cry protein yields were analyzed with Imagen Lab 5.1 program (BioRad Hercules CA, USA). For Western blot analysis, proteins were electroblotted onto Amersham Hybondmembrane (Amersham Biosciences, Little Chanfont, UK) for 2 h at 20 V using a semi-dry

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ACCEPTED MANUSCRIPT blotter (C.B.S. Scientific Co., Del Mar CA, USA). The membrane was blocked for 2 h with 5% (w/v) non-fat milk in PBS (pH 7.4) and incubated for 2 h with polyclonal antibodies raised against ChiA74∆sp. Finally, membranes were washed with 20 mM Tris-HCl pH 7.5, 160 mM NaCl, incubated for 1 h with anti-rabbit alkaline phosphatase and developed with

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NBT/BCIP according to manufacturer’s protocol (Roche, Mannheim, Germany).

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2.8. Viable spores count

Recombinant and wild type bacteria were grown in NB at 28°C, 200 rpm until

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>95% of cells sporulated and lysed (~96 h). Then 100 μL of each culture was collected and

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incubated at 60°C for 20 min to destroy the remaining vegetative cells. Serial dilutions of 10-5-10-7 of the suspension were plated onto Nutrient agar (NA) and cultures were

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incubated at 28°C for 24 h to determine the number of viable spores. Data were analyzed

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with the ANOVA program (StatSoft Inc., Tulsa, USA). Experiments were performed in

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triplicate 12.

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2.8. Phase contrast and scanning electron microscopy Bacteria were cultured in NB at 28ºC, 200 rpm until >95% of cells had sporulated

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and lysed (~96 h), as determined by phase contrast microscopy. The area of bipyramidal crystals in wild type and recombinant strains were estimated using the AxioVision LE program (Carl Zeiss Microscopy, Oberkochen, Germany). Additionally, cultures were centrifuge and supernatants were discarded to eliminate secreted molecules such as putative antimicrobial peptides, proteases, chitinases and Vip proteins. Pellets (spore/crystal of HD1, and spore/crystals/putative ChiA74∆sp inclusions of recombinant HD-1) were

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ACCEPTED MANUSCRIPT washed 3x with double distilled water, and lyophilized. Powders were covered with gold in a JEOL fine-coat ion sputter (model JFC-1100) for the scanning electron microscopy (SEM) analysis with Field Emission Scanning Electron Microscope, Carl Zeiss, model Sigma HD-VP (Oberkochen, Germany). The images were obtained using an Angle

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selective Backscatter detector (AsB), under High-Vacuum (HV) conditions with a X-15kV

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beam.

2.9 Bioassays

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A colony of Spodoptera frugiperda was maintained on artificial diet at 25 ± 2 ºC

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and 80 ± 10% relative humidity, under a 16:8 h (light:dark) photoperiod. Six different concentration of lyophilized samples suspended in tap water and ranging from 26.03 to 833

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ng/cm2 were used in bioassays; tap water was used as the negative control. Bioassays were

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performed in triplicate. A constant volume of 500 L of each concentration was applied onto the surface of diet contained in Petri dishes (area 60 cm2). Twenty larvae, 24 h after

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hatching, were used to test each concentration. Mortalities were recorded after 5 days of

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incubation. The mean concentration at which 50% (LC50) of the larvae died was estimated

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by Probit analysis 18.

3. Results 3.1 Differential expression from chiA-p, BtI/BtII-p and cyt-p/STAB-SD promoters We compared the expression constructs with chiA-p, BtI/BtII-p and cyt-p/STAB-SD, promoters that regulate expression of chiA74, cry and cyt genes in B. thuringiensis,

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ACCEPTED MANUSCRIPT respectively, by measuring the fluorescence emitted by the GFP or the enzymatic activity of ChiA74 using a fluorescent substrate (Fig.1). Results of both assays confirmed that the chiA-p promoter was active at ~4 h during the lag phase, and expression continued throughout the exponential, stationary to the death phase. However, expression driven by

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BtI/BtII-p and cyt-p/STAB-SD occurred in the stationary through the death phase. The data

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confirmed that chiA74-p and BtI/BtII-p, cyt-p/STAB-SD promoters were active during

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vegetative and sporulation phases, respectively, as previously reported 14,19. The highest

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level of expression occurred with cyt-p/STAB-SD followed by BtI/BtII-p and chiA74-p (Fig.

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1).

3.2. Recombinant strains harboring truncated chiA74∆sp under control of chiA-p

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Based on its lower level of expression detected during vegetative growth we

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decided to use chiA-p instead of the strong cyt-p/STAB-SD to drive expression of truncated chiA74∆sp (Fig. 2A) harbored in a low copy number (~ 4 copies per chromosome)

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plasmids, pHT3101, 20 in HD1 and Cry-B. To confirm the presence of the recombinant

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plasmids in HD1 and Cry-B transformants, the erythromycin resistance gene ( 1.2 kbp)

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harbored in the pHT3101 shuttle vector, and the three different chiA74 versions (i.e. chiA74Δsp, chiA74Δsp-60, chiA74Δsp-50) were amplified by PCR and sequenced. As was expected, the erythromycin resistance gene was amplified in all recombinant strains including HD1 transformed with the pHT3101 parental vector (Fig. 2B); no amplicons were observed with the wild type HD1 or Cry-B controls. The mature (chiA74Δsp), and truncated versions (chiA74Δsp-60, chiA74Δsp-50) were also amplified from the recombinant strains obtaining amplicons of  2.0, 1.5 and 1.3 kbp, respectively (Fig. 2B).

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3.3. Truncated versions of ChiA74 synthesized in B. thuringiensis have enzymatic activity Because all chitinase constructs lack the secretion signal peptide coding sequence it was expected that the recombinant strains would produce chitinase inclusions in B.

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thuringiensis and that these might be released together with spores and bipyramidal crystals

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after autolysis. As such, from lysed cultures, supernatants were discarded and the pellets

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containing spores, crystals, and putative IBs were used to determine chitinase activity using 4-MU-GlcNAc3 (synthetic fluorogenic substrate) and colloidal chitin (natural substrate)

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(Table 2). Enzymatic activity was not detected in preparation of Cry-B and HD1 parental

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strains. In contrast, chitinase activity was detected in preparations from pellets of recombinant strains producing ChiA74∆sp and its truncated versions. Cry-B transformed

showed

higher

activity followed

by HD1/ChiA74∆sp-60

and

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HD1/ChiA74∆sp

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with the different constructs showed activities of 5 U/mg without significant differences.

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HD1/ChiA74∆sp-50, with values of  16 U/mg, 11 U/mg and  6 U/mg, respectively. With colloidal chitin, we did not observe significant differences in the activities of

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HD1/ChiA74∆sp and HD1/ChiA74∆sp-50 (i.e. 4.5 U/mg), nor between CryB/ChiA74∆sp and Cry-B/ChiA74∆sp-50 (i.e. 2.3 U/mg); these recombinant strains

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showed higher activities of 38% and 35% when compared with HD1/ChiA74∆sp-60 and Cry-B/ChiA74∆sp-60, respectively. The data indicated that mature ChiA74 expressed in recombinant B. thuringiensis strains had similar activity as ChiA74∆sp-50 with colloidal chitin as the substrate. Similar results were observed with ChiA74∆sp and its truncated versions synthesized in E. coli 15. Lastly, chitinase activity of recombinants HD1 strains

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ACCEPTED MANUSCRIPT showed increases from ~ 49 to 128-fold and ~7 to 12-fold, compared with parental HD1, respectively, when 4-MU-GlcNAc3 and colloidal chitin were used as substrates.

3.4. Phase contrast and scanning electron microscopy, viable spore number and Cry

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protein yields

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The area of bipyramidal crystals produced by HD1 (i.e.  1.64 µm2) was similar to

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those produced in HD1/ChiA74∆sp-60 and HD1/ChiA74∆sp-50, but higher than

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HD1/ChiA74∆sp (1.52 µm2) (Table 3). These data suggested that the truncated chitinase genes expressed under chiA-p could be used for production of variants of active ChiA74

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with little or no detrimental effects on native Cry1A crystal formation. Similar data were obtained when volumes of the crystal produced in HD1 and its recombinants were

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estimated from scanning electron micrographs; all bipyramidal crystals had similar volumes

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( 0.08 µm3) with no significant differences.

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We note that SEM analysis of lysed samples of HD1 and its recombinants revealed the typical bipyramidal crystals. In some fields it was possible to distinguish the typical

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cuboidal inclusion associated to the bipyramidal crystals (Fig. 3, panels I, II). The bipyramidal crystals of HD1 are formed by Cry1Aa, Cry1Ab and Cry1Ac which have

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molecular masses of 120-130 kDa, whereas the cuboidal inclusions are composed Cry2A proteins of 60 kDa (Fig. 3, panel III) 1. When crystals were carefully analyzed by scanning electron microscopy, it was observed that the surface of the bipyramidal crystals in the recombinant strains were rougher than HD1 (Fig. 3, panel II). We do not have a clear explanation for this phenomenon, and we hypothesize that the rugosity could be due to the presence of putative amorphous small IBs of ChiA74∆sp or its truncated versions attached

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ACCEPTED MANUSCRIPT to the surface or embedded in the crystals. Western blot analysis using polyclonal antiChiA74∆sp showed a weak signal with proteins of 50 kDa in recombinant strains indicating that presence of processed ChiA74∆sp (Fig. 3, panel IV), as observed elsewhere 21.

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With regard to spore counts, recombinant strains showed an increase in viable spores of

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~46% and 66% compared to wild type HD1 and non-transformed Cry-B, respectively.

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Finally, the yield of Cry1A per unit medium produced by HD1 and recombinants was analyzed using 10% SDS-PAGE. HD1/ChiA74∆sp-60 synthesized ~ 20% more Cry1

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compared to HD1, and HD1/ChiA74∆sp-50 produced ~ 40% more of the crystal toxin

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3.6 Bioassays against S. frigiperda

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when compared to HD1/ChiA74∆sp (Fig. 3, panel III).

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When spore/Cry crystal mixtures of HD-1 and spore/Cry crystal/ChiA74sp

wildtype

HD1

was

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inclusion body mixtures of recombinants were assayed against S. frugiperda the LC50 for 185.65

ng/cm2.

HD1/ChiA74∆sp,

HD1/ChiA74∆sp-60

and

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HD1/ChiA74∆sp-50 showed LC50s of 167.58 ng/cm2,117.11 ng/cm2 and 133.32 ng/cm2,

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respectively, which represent an apparent 1.1x, 1.58x, 1.39x enhancement in the toxicity of the recombinant strains. Unfortunately, we did not detect a significant difference in the LC50s as there was an overlap between the upper fiducial limit of the recombinant strain’s LC50 and the lower fiducial limit of the LC50 of the wildtype. Reduction of 10%, 37% and 38% of the amount (ng/cm2) required for HD1/ChiA74∆sp, HD1/ChiA74∆sp-60 and HD1/ChiA74∆sp-50, respectively, indicated that recombinants strains required lower concentration than the wildtype (HD1) to kill 50% of the S. frugiperda larvae (Table 4).

15

ACCEPTED MANUSCRIPT

4. Discussion The selection of an appropriate promoter for heterologous expression is an important factor for production of recombinant proteins in B. thuringiensis 19,21. We

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decided to use a promoter that did not compete with BtI/BtII, sporulation-dependent

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promoters that drive expression of different cry genes, including those present in HD1 (i.e.

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cry1A, cry2A) 22. As such, the promoter chiA-p is activated in the late lag phase (~ 4 h)

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and continues to ~96 h, with a peak activity occurring within ~36 h. Similar expression has been observed with a chitinase promoter from B. thuringiensis subsp. israelensis 75 for

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which expression begins at ~6 h and peaks at 60 h 23. Although chiA-p expression overlapped with BtI/BtII-p and cyt-p/STAB-SD during the stationary phase at ~36 h, the

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level of activity was significantly lower when compared with these sporulation-dependent

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promoters which are activated by different factors. The chiA-p sequence contains only

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one promoter that is likely activated by A during the vegetative growth 12,14,23,24. In contrast, BtI/BtII-p and cyt-p/STAB-SD harbor two and three promoters (BtI/BtII/BtIII-p),

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respectively, recognized by E and  factors that are maximally activated in the stationary

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and sporulation phases of growth 19,22,25. Using chiA-p we showed that lysed samples of recombinant HD1 were composed of spores/crystals/IBs and had higher chitinase activities than parental HD1 suggesting that the engineered chitinases were fully or partially folded, at least in the constraints of our assay parameters. In general, ChiA74∆sp and its truncated versions did not have significant differences in the activity against 4-MU-(GlcNAc)3. With colloidal chitin, ChiA74Δsp and ChiA74Δsp-50, the enzyme version lacking the chitin binding domain (CBD) and

16

ACCEPTED MANUSCRIPT fibronectin-like domain (FnIII), have similar activity, but higher than that of ChiA74Δsp60, suggesting that the CBD and FIII are dispensable for activity against colloidal chitin as previously reported 15. A similar behavior has been observed with a chitinase from V. harveyi 26.

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Importantly, our objective was to produce active chitinases while maintaining the

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native structural integrity of Cry1A crystals in recombinant HD1, unlike that of our

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previous studies showing expression of chiA74∆sp using pytA-p/STAB-SD resulted in a

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significant reduction (~30%) in size of HD1’s bipyramidal crystal 12. Indeed, using chiAp to drive expression of the chitinase gene constructs negligible or no effect on the size of

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the bipyramidal crystals in HD1 was observed (Table 3). Nevertheless, although we obtained increased enzymatic activities, for example with the recombinant ChiA74Δsp, a

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reduction in the amount of Cry1A synthesized per unit culture medium of 20% relative to

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a strain HD1 (Table 2, Fig. 3, panel III) was observed. Similar results have been

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documented elsewhere 27. Interestingly, HD1/ChiA74Δsp-60 which had lower activity against colloidal chitin, showed an increased Cry1A yield of 20% and 40% relative to

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HD1 and HD1/ChiA74Δsp, respectively, and was the most toxic of the recombinants

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requiring 117 ng/cm2 diet to kill 50% of S. frugiperda. It is likely that the higher number of viable spores of HD1/ChiA74Δsp-60 per unit medium compared with HD1 is in part responsible for increased Cry1A yield and larvicidal activity. Comparing the activities reported in our previous and present studies, we note that samples containing ChiA74∆sp expressed with chiA-p in the present study (14 U/mL) was 9x higher than the activity of ChiA74∆sp ( 1.5 U/mL) when the gene was expressed with cytA-p/STAB-SD in HD1, at pH 6.8 12. This indicates that ChiA74∆sp expressed under

17

ACCEPTED MANUSCRIPT regulation of a vegetative promoter (chiA-p) results in synthesis of more active chitinase than when expression is regulated by a strong sporulation-dependent promoter. The amorphous IBs present on the surface of purified bipyramidal crystals of recombinants (Fig. 3), which were not observed in HD1, taken together with increased

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chitinase activities in these preparations suggested that the IBs were aggregates of the

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chitinases. However, the weak Western blot signals at ~50 kDa indicates that intracellular

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ChiA74∆sp and ChiA74∆sp-60 are unstable and are processed to a small active protein,

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similar to that reported for other chitinases of B. thuringiensis 11,21. We cannot discard the possibility that these IBs also contain native chitinases and/or other cellular components

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whose composition can vary depending on pH and growth temperature 28,29. Finally, although increases in toxicity of 10%, 37% and 29% of HD1/ ChiA74∆sp,

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HD1/ ChiA74∆sp-60 and HD1/ChiA74∆sp-50, respectively, relative to HD1, against S.

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frugiperda were observed, the differences were not statistically significant. It is possible

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that the contributions of the ChiA74 chitinase variants to toxicity could be limited by their sub-optimal levels of synthesis and proper folding, and sub-optimal solubility of their

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5. Conclusions

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corresponding IBs.

Our study shows that chiA74∆sp, chiA74∆sp-60 and chiA74∆sp-50 under the control of chiA-p expressed in B. thuringiensis HD1 resulted in the synthesis of putative amorphous inclusion bodies (IBs) composed of the corresponding enzyme. Importantly, intracellular accumulation of these IBs did not affect the size of the bipyramidal crystals and yield of Cry1A per unit of culture medium. Bioassays against S. frugiperda using sporulated/lysed

18

ACCEPTED MANUSCRIPT samples of the recombinant strains compared with HD1 did not show statistically significant differences in LC50s. Nevertheless, we have demonstrated that our system is useful for producing intracellular chitinases in B. thuringiensis as it does not affect the size

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of the native bipyramidal crystals.

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Competing interests

The authors report no conflicts of interest in this work. The authors declared no competing

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financial interest.

Acknowledgements

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This research was supported by Grant SEP-CONACyT (258220) México, to J.E. Barboza-

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Corona. Karen S. González-Ponce is a doctoral student in the graduate program in BioSciences of the University of Guanajuato (UG), México and is supported by a

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scholarship from CONACyT. L.E. Casados-Vázquez is a Young Associate Researcher

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supported by Grant Cátedras-CONACyT (2069). We appreciate the help of Dr. Ricardo Navarro from the University of Guanajuato CONACYT National Laboratory for SEM-

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EDX microphotographs, and the technical assistance of Isamar Sánchez-Vargas, Samuel Celaya and Jonathan Rangel from the University of Guanajuato.

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Galván, D.K. Bideshi, J.E. Barboza-Corona, High-level synthesis of endochitinase

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ACCEPTED MANUSCRIPT [7] X. Ding, Z. Luo, B. Gao, Y. Sun, Y. Zhang, Improving the insecticidal activity by expression of a recombinant cry1Ac gene with chitinase-encoding gene in acrystalliferous Bacillus thuringiensis, Curr. Microbiol. 56 (2008) 442-446. [8] J.E. Barboza-Corona, T. Ortiz-Rodríguez, N.M. de la Fuente-Salcido, J. Ibarra, D.K.

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[12] J.E. Barboza-Corona, J.L. Delgadillo-Ángeles, J.C. Castañeda-Ramíırez, U.E. Barboza-Pérez, L.E. Casados-Vázquez, D.K. Bideshi, M.C. del Rincón Castro, Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74∆sp chitinase inclusions, Cry crystals and spores for applied use, Microb. Cell Fact. 13 (2014) 15.

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ACCEPTED MANUSCRIPT [13] K.S. González-Ponce, L.E. Casados-Vázquez, R. Salcedo-Hernández, D.K. Bideshi, M.C. del Rincón Castro, J.E. Barboza-Corona, Recombinant Bacillus thuringiensis subsp. kurstaki HD73 strain that synthesizes Cry1Ac andchimeric ChiA74∆sp chitinase inclusions, Arch. Microbiol. 199 (2017) 627-633.

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[14] J.E. Barboza-Corona, E. Nieto-Mazzocco, R. Velázquez-Robledo, R. Salcedo-

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the chitinase gene chiA74 from Bacillus thuringiensis, Appl. Environ. Microbiol. 69 (2003) 1023–1029.

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[15] E.O. Juárez-Hernández, L.E. Casados-Vázquez, D.K. Bideshi, R. Salcedo-Hernández,

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J.E. Barboza-Corona, Role of the C-terminal and chitin insertion domains on enzymatic activity of endochitinase ChiA74 of Bacillus thuringiensis, Int. J. Biol. Macromolec. 102

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(2017) 52-59.

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[16] L.E. Casados‐Vázquez, D.K. Bideshi, J.E. Barboza‐Corona, The thnR gene is a negative transcription regulator of the thurincin H genetic cassette in Bacillus

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thuringiensis subsp. morrisoni, Arch. Microbiol. 199 (2017) 385-390.

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[17] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head bacteriophage T4, Nature 227 (1970) 680-685.

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[18] J.C. Rangel-Núñez, M.F. Vázquez-Ramírez, M.C. del Rincón-Castro, Biological and molecular characterization of exotic baculovirus strains with activity to mexican population of fall armyworm Spodoptera frugiperda (Lepidóptera: Noctuidae), Interciencia 39 (2014) 320-326. [19] Y. Sakano, H.W. Park, D.K. Bideshi, G. Baoxue, B.A. Federici, Contributions of 5´UTR and 3´-UTR cis elements to Cyt1Aa synthesis in Bacillus thuringiensis subsp. israelensis, J. Invertebr. Pathol. 149 (2017) 66-75. 22

ACCEPTED MANUSCRIPT [20] D. Lereclus, O. Arantés, J. Chaufaux, N.M. Lecadet, Transformation and expression of a cloned δ-endotoxin gene in Bacillus thuringiensis, FEMS Microbiol. Lett. 60 (1989) 211-218. [21] L.E. Casados‐Vázquez, S. Avila-Cabrera, D.K. Bideshi, J.E. Barboza‐Corona,

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Heterologous expression, purification and biochemical characterization of endochitinase

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ChiA74 from Bacillus thuringiensis, Protein Expr. Purif. 109 (2015) 99-105.

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[21] H.W. Park, R.H. Hice, B.A. Federici, Effect of promoter and plasmid copy number on Cyt1A synthesis and crystal assemby in Bacillus thuringiensis, Curr. Microbiol. 72

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(2016) 33-40.

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[22] C. Deng, Q. Peng, F. Song, D. Lereclus, Regulation of cry gene expression in Bacillus thuringiensis, Toxins 6 (2014) 2194-2209.

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[23] C.C. Xie, Y. Luo, Y.H. Chen, J. Cai, Construction of a promoter-probe vector for

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Bacillus thuringiensis: the identification of cis-acting elements of the chiA locus, Current. Microbiol. 64 (2012) 492-500.

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[24] H. Agaisse, D. Lereclus, How does Bacillus thuringiensis produce so much

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insecticidal crystal protein? J. Bacteriol. 177 (1995) 6027-6032. [25] A. Bravo, H. Agaisse, S. Salamitou, D. Lereclus, Analysis of cryIAa expression in

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sigE and sigK mutants of Bacillus thuringiensis, Mol. Gen. Genet. 250 (1996) 734-741. [26] W. Suginta, P. Sirimontree, N. Sritho, T. Ohnuma, T. Fukamizo, The chitin-binding domain of a GH-18 chitinase from Vibrio harveyi is crucial for chitin-chitinase interactions, Int. J. Biol. Macromol. 93 (PtA) (2016) 1111-1117. [27] G. Casique-Arroyo, D.K. Bideshi, R. Salcedo-Hernández, J.E. Barboza-Corona, Development of a recombinant strain of Bacillus thuringiensis subsp. kurstaki HD-73 that produces the endochitinase ChiA74, Antonie van Leeuwenhoek 92(1) (2007)1-9. 23

ACCEPTED MANUSCRIPT [28] N.S. De Groot, S. Ventura, Effect of temperature on protein quality in bacterial inclusion bodies, FEBS Lett. 580 (2006) 6471-6476. [29] A. Castellanos-Mendoza, R.M. Castro-Acosta, A. Olvera, G. Zavala, B. MendozaVera, E. García-Hernández, A. Alagón, M.A. Trujillo-Roldán, N.A.Valdez- Cruz,

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Influence of pH control in the formation of inclusion bodies during production of

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recombinant sphingomyelinase-D in Escherichia coli, Microb. Cell Fact. 13 (2014) 137.

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Figure legends Fig. 1. Recombinant gene constructs and time of promoter´s expression. (A) Promoters chiA-p, BtI/BtII-p and cyt-p/STAB-SD were ligated to chimeric construct chiA74∆sp∆tt-gfp in the pHT3101 shuttle vector. Arrows indicated transcription direction; RBS, ribosome

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T

binding site; T, transcriptional terminator. (B) Growth curve of Cry-B cultivated in nutrient

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broth. (C) Assessment of temporal expression from different promoters by measuring the fluorescence emitted by GFP. Constructs in (A) were transformed into B. thuringiensis Cry-

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B to obtain chiA-p-chiA74ΔspΔtt-gfp/Cry-B, BtI/BtII-p-chiA74ΔspΔtt-gfp/Cry-B and cytp/STAB-SD-chiA74ΔspΔtt-gfp/Cry-B. Cry-B, is a nontransformed bacterium; pHT3101

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indicates that this bacterium was transformed with plasmid pHT3101. (B) Temporal

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expression of the different promoters assessed by measuring fluorescence emitted by 4-

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MU-(GlcNAc)3 following hydrolysis by ChiA74∆sp.

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Fig. 2. Structural organization of mature and truncated recombinant ChiA74 expressed in B. thuringiensis. (A) CD, catalytic domain; CID, chitin insertion domain; FnIII,

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fibronectin-like domain; CBD, chitin binding domain; RBS, ribosome binding site; chiA-p,

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wild promoter of chiA74; T, transcriptional terminator. Oligonucleotides (Table 1) used to generate the different constructs. All constructs were ligated into the pHT3101 vector that was used to transform B. thuringiensis HD1 and Cry-B. (B) Confirmation of the transformation of HD1 by PCR. Lane 1, 1 kb (kbp, kilobase pairs) DNA ladder lanes 2, HD1/pHT3101; lanes 3 and 4, HD-1; lanes 5, and 6, HD-1/ChiA74∆sp; lane 7 and 8, HD1/ChiA74∆sp-60; lanes 9, and 10, HD-1/ChiA74∆sp-50. Amplicons of lanes 2, 4, 6, 8, 10 were obtained using the primers ery-1 and ery-2 to amplify the erythromycin resistance

25

ACCEPTED MANUSCRIPT gene harbored in the pHT3101. Amplicons in lanes 3 and 5, lane 7 and lane 9 were amplified using the primers N-termchiA/C-termchiA, N-termchiA/CAT/FnIII and NtermchiA/CAT/CID to obtain chiA74∆sp, chiA74∆sp-60 and chiA74∆sp-50, respectively.

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Similar results were obtained with Cry-B and its recombinants.

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Fig. 3. Scanning electron microscopy (SEM) and protein analysis. Panel I, SEM of samples

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observed in a JEOL fine-coat ion sputter, model JFC-1100 electronic microscope. (a) HD1 wild type, (b) HD1/ChiA74∆sp, (c) HD1/ChiA74∆sp-60, (d) HD1/ChiA74∆sp-50. Panel II,

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one crystal from each strain (panel I) was taken and amplified. The surface of the

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bipyramidal crystals in the recombinant strains (c, d, and less in e) appear to be rougher than those of HD1. The rugosity might be due for the presence of ChiA74∆sp and its

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truncated versions which are embedded in the crystals. Panel III, 10% SDS-PAGE. (M)

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Page ruler unstained protein ladder (ThermoFisher Scientific), (a) HD1 wild type, (b) HD1/ChiA74∆sp, (c) HD1/ChiA74∆sp-60, (d) HD1/ChiA74∆sp-50. Asterisks indicated the

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position of proteins of  120 and 60 kDa that correspond to Cry1 and Cry2 proteins which

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are assembled to make bipyramidal (bip) and cuboidal (cub) inclusions, respectively. Ratio corresponding to Cry1 protein yield was analyzed with Imagen Lab 5.1 program (BioRad

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Hercules CA, USA). Panel IV, Western blot analysis using an anti-ChiA74∆sp antibody; (a) HD1 wild type, (b) HD1/ChiA74∆sp, (c) HD1/ChiA74∆sp-60, (d) ChiA74∆sp expressed in E. coli and purified by Ni affinity column used as positive control. Arrows at the right or left indicate the position of ChiA74∆sp or its putative processed form of 50 kDa, respectively.

26

ACCEPTED MANUSCRIPT

8.000

chiA∆ps∆tt

RBS

gfp

7.000 6.000

RBS

gfp

5.000 4.000 3.000

T

chiA∆ps∆tt

O.D. 600nm

BtI/BtII-p

2.000

cytA-p/STAB-SD

0.000

gfp

0

20

30

40

50

60

70

80

90

T(h)

D

160000 140000

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120000 100000

RFU

10

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1.000

chiA∆ps∆tt

RBS

C

Cry-B

B

chiA-p

CR

A

80000

40000 20000 0 4

8

12

24 36 48 60 72 T (h) cyt-p/STAB-SD BtI/Bt II-p Cry-B

84 pHT3101

96

M

chiA-p

AN

60000

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Fig. 1. Recombinant gene constructs and time of promoter´s expression. (A) Promoters chiA-p, BtI/BtII-p and cyt-p/STAB-SD were ligated to chimeric construct chiA74∆sp∆tt-gfp

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in the pHT3101 shuttle vector. Arrows indicated transcription direction; RBS, ribosome binding site; T, transcriptional terminator. (B) Growth curve of Cry-B cultivated in nutrient

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broth. (C) Assessment of temporal expression from different promoters by measuring the fluorescence emitted by GFP. Constructs in (A) were transformed into B. thuringiensis Cry-

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B to obtain chiA-p-chiA74ΔspΔtt-gfp/Cry-B, BtI/BtII-p-chiA74ΔspΔtt-gfp/Cry-B and cytp/STAB-SD-chiA74ΔspΔtt-gfp/Cry-B. Cry-B, is a nontransformed bacterium; pHT3101 indicates that this bacterium was transformed with plasmid pHT3101. (B) Temporal expression of the different promoters assessed by measuring fluorescence emitted by 4MU-(GlcNAc)3 following hydrolysis by ChiA74∆sp.

27

ACCEPTED MANUSCRIPT (A)

Recombinant strains

N-termchiA chiA-p

pHT3101

CD

RBS

CID

FnIII

HD1/ or Cry-B/chiA74Dsp

CBD C-termchiA

N-termchiA chiA-p

CD

RBS

pHT3101

CID

FnIII

HD1/ or Cry-B/chiA74Dsp-60

CAT/FnIII

N-termchiA

CD

RBS

CID

HD1/ or Cry-B/chiA74Dsp-50

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pHT3101

T

chiA-p

(B)

1

2

3

4

5

6

7

8

9

CR

CAT/CID

10

Kbp 3.0

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2.0 1.5 1.0

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0.7

Fig. 2. Structural organization of mature and truncated recombinant ChiA74 expressed in

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B. thuringiensis. (A) CD, catalytic domain; CID, chitin insertion domain; FnIII, fibronectin-like domain; CBD, chitin binding domain; RBS, ribosome binding site; chiA-p,

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wild promoter of chiA74; T, transcriptional terminator. Oligonucleotides (Table 1) used to generate the different constructs. All constructs were ligated into the pHT3101 vector that

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was used to transform B. thuringiensis HD1 and Cry-B. (B) Confirmation of the transformation of HD1 by PCR. Lane 1, 1 kb (kbp, kilobase pairs) DNA ladder; lanes 2,

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HD1/pHT3101; lanes 3 and 4, HD-1; lanes 5, and 6, HD-1/ChiA74∆sp; lane 7 and 8, HD1/ChiA74∆sp-60; lanes 9, and 10, HD-1/ChiA74∆sp-50. Amplicons of lanes 2, 4, 6, 8, 10

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were obtained using the primers ery-1 and ery-2 to amplify the erythromycin resistance gene harbored in the pHT3101. Amplicons in lanes 3 and 5, lane 7 and lane 9 were amplified using the primers N-termchiA/C-termchiA, N-termchiA/CAT/FnIII and NtermchiA/CAT/CID to obtain chiA74∆sp, chiA74∆sp-60 and chiA74∆sp-50, respectively. Similar results were obtained with Cry-B and its recombinants.

28

ACCEPTED MANUSCRIPT I

III b

a

kDa

M

a

b

c

d

200 150 120 100 85

bip

* Cry1, bip

70 60

** Cry2, cub

50

cub

T

40

d

IP

30

c

25

a

1.0

0.8

b

c

1.2

1.0

* Ratio Cry1

d

II a

b

AN

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IV

CR

20

c

d

ED

M

c

AC

CE

PT

Fig. 3. Scanning electron microscopy (SEM) and protein analysis. Panel I, SEM of samples observed in a JEOL fine-coat ion sputter, model JFC-1100 electronic microscope. (a) HD1 wild type, (b) HD1/ChiA74∆sp, (c) HD1/ChiA74∆sp-60, (d) HD1/ChiA74∆sp-50. Panel II, one crystal from each strain (panel I) was taken and amplified. The surface of the bipyramidal crystals in the recombinant strains (c, d, and less in e) appear to be rougher than those of HD1. The rugosity might be due for the presence of ChiA74∆sp and its truncated versions which are embedded in the crystals. Panel III, 10% SDS-PAGE. (M) Page ruler unstained protein ladder (ThermoFisher Scientific), (a) HD1 wild type, (b) HD1/ChiA74∆sp, (c) HD1/ChiA74∆sp-60, (d) HD1/ChiA74∆sp-50. Asterisks indicated the position of proteins of  120 and 60 kDa that correspond to Cry1 and Cry2 proteins which are assembled to make bipyramidal (bip) and cuboidal (cub) inclusions, respectively. Ratio corresponding to Cry1 protein yield was analyzed with Imagen Lab 5.1 program (BioRad Hercules CA, USA). Panel IV, Western blot analysis using an anti-ChiA74∆sp antibody; (a) HD1 wild type, (b) HD1/ChiA74∆sp, (c) HD1/ChiA74∆sp-60, (d) ChiA74∆sp expressed in E. coli and purified by Ni affinity column used as positive control. Arrows at the right or left indicate the position of ChiA74∆sp or its putative processed form of 50 kDa, respectively.

29

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Table 1 Primers used for PCR amplification of different promoters and open reading frames Sequence a

Primer

D:5’- CAAAACTGCAGGTTTTCGCTAATGACGGCATTTA-3’

GFP-RW

R:5’- CAAAACTGCAGTTATTTGTAGAGCTCATCCAT-3’

BtI-II-1

D:5’-CCGGAATTCTTACAATTCAAGATGAATTGCAGG-3’

BtI-II-2

R: 5’- CAAAACTGCAGAAGTTACCTCCATCTCTTTTATTAAG-3’

Cyt/stab-1

D: 5’-CGGAATTCTATTTTCGATTTCAAATTTTCC-3’

Cyt/stab-2

R: 5’-CAAAACTGCAGTTCTCCTTTCAAATAAAAGATAT-3’

chiA-1

R: 5’ -CGGAATTCCTTTAATATATCTTTTTGTAGTTCCA-3’

chiA2

F: 5’ -CAAAACTGCAGTTCTCCTTTCAAAATAAAAGATA-3’

AN

US

CR

IP

T

ChiA74-1

M

N-termchiA F: 5’- AACTGCAGGATTCACCAAAGCAAAGTCAAAAA -3’

ED

C- termchiA R: 5’- AACTGCAGCTTGTCGAATTTTTTCTTCAGCA-3’ R: 5’- AACTGCAGCTCAGTATCTTTTTGATTAATAGGTCC -3’

CAT/FnIII

R: 5’- AACTGCAGATTAGCTTCATCCGTTTTGACAGTAAGAGC -3’

Termchi-1

F: 5’- ACATGCATGCATTTAGGTTTGAGCAATACCTC-3’

Termchi-2

R: 5’- ACATGCATGCCGAAAGCCTTTCCCTAACAGG-3’

ery-1

F: 5’-AAAACTGCAGCTTAAGAGTGTGTTGATAGTGC-3’

a

CE

AC

ery-2

PT

CAT/CID

R: 5’-ATAAGAATGCGGCCGCCCCCGTAGGCGCTAGGGACC-3’

D and F, direct and forward primers, respectively. The restriction endonuclease cleavage

site for EcoRI, PstI, SphI and NotI are underlined.

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Table 2 Chitinase activity of recombinant strains of Bacillus thuringiensis harboring mature and truncated versions of ChiA74∆sp using different substrates 4-MU-GlcNAc3* U/mg

Colloidal chitin**

U/mL

U/mg

T

Strains

0.124±0.10a

0.47±0.09a

0.41±0.10a

HD1/ChiA74∆sp

15.946±1.74b

14.58±2.35b

HD1/ChiA74∆sp-60

10.86±0.817c

10.01±1.89c

3.0±0.8c

HD1/ChiA74∆sp-50

6.085±1.887d

8.63±0.95c

4.63±0.9b

Cry-B

0.106±0.02a

0.61±0.06a

0.19±0.2a

Cry-B/ChiA74∆sp

5.64±0.09b

5.89±0.32b

Cry-B/ChiA74∆sp-60

5.27±0.477b

Cry-B/ChiA74∆sp-50

5.716±0.312b

4.85±0.05b

2.3±0.02b

5.39±0.28b

1.43±0.1c

5.90±0.43b

2.26±0.5b

M

AN

US

CR

IP

HD1 wild type

ED

*One Unit of activity was defined as the amount of enzyme required to release 1 mol of MU in 1h.

PT

**One Unit of activity was defined as the amount of enzyme required to release 1 mol of NAG in 1h. Different letter in the same column (a, b) indicate significant difference at

AC

CE

P<0.05 determined by the Tukey multiple range test.

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Table 3 Comparison of crystal area*, volume** and viable spores in the parental and recombinants strains of B. thuringiensis expressing

T P

ChiA74

I R

Crystal area µm2

Ratio

Length (µm)

Width (µm)

Vol (µm3)

1.64±0.383a***

1.07

0.690 ± 0.150

0.400 ± 0.080

HD1/ChiA74∆sp

1.52±0.363b

1.0

0.580 ± 0.105

0.430 ± 0.080

HD1/ChiA74∆sp-60

1.64±0.338a

1.07

0.600 ± 0.125

0.430 ± 0.075

HD1/ChiA74∆sp-50

a

Strains HD1

1.64±0.305

1.07

0.620 ± 0.070

NPC

Cry-B

NPC****

-

Cry-B/ChiA74∆sp

NPC

-

Cry-B/ChiA74∆sp-60

NPC

-

Cry-B/ChiA74∆sp-50

NPC

T P

D E

-

NPC NPC NPC

M

A

Spore/mL x 106

Ratio

0.078 ± 0.040a

1.00

60±0.6a

1.00

0.077 ± 0.035a

0.98

86±1.0b

1.43

0.082 ± 0.048a

1.05

89±1.5b

1.48

1.01

88±1.0

b

1.46

C S

U N

0.415 ± 0.170

Ratio

0.079 ± 0.040

a

NPC

NPC

-

56±0.6a

1.00

NPC

NPC

-

91±2.0b

1.62

NPC

NPC

-

93±2.5b

1.66

-

b

1.69

NPC

NPC

95±1.0

E C

* Crystal areas were estimated using the AxioVision LE program (Carl Zeiss Microscopy, Göttingen Germany). ** Crystal volumes were estimated measuring the length and width of crystal using scanning electron micrograph. *** Different lower letter in the same column (a, b) indicate significant difference at P<0.05 determined by the Tukey multiple range test. ****NPC, not produce crystals.

C A

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TABLE 4 Statistical parameters for estimating the LC50 of strains of B. thuringiensis against fall armyworm Spodoptera frugiperda LC50**

Slope

X2

HD1

185.652 (112.475-306.437)

1.285

2.642

HD1/ChiA74∆sp

167.598 (108.147-259.731)

1.603

HD1/ChiA74∆sp-60

117.115 (62.728-218.656)

1.001

1.730

HD1/ChiA74∆sp-50

133.321 (79.025-224.922)

1.228

1.097

*HD1,

were

HD1

transformed

IP

1.109

CR

US

and

T

Strains*

with

chiA74Δsp

(HD1/ChiA74∆sp)

and

AN

chiA74ΔspΔCBD (HD1/ChiA74∆sp-60) and chiA74ΔspΔCBDΔFnIII (HD1/ChiA74∆sp-

M

50). **

Values are shown in nanograms per cm2 of mixtures of spore/crystal mixture or

ED

spore/crystals/IBs of HD1 or recombinant strains, respectively, and represent 5-days

AC

CE

PT

mortality as determined by Probit analysis. Fiducial limits are indicated in parenthesis.

33