Bacillus thuringiensis subsp. kurstaki HD1 as a factory to synthesize alkali-labile ChiA74∆sp chitinase inclusions, Cry crystals and spores for applied use.
BACKGROUND
The endochitinase ChiA74 is a soluble secreted enzyme produced by Bacillus thuringiensis that synergizes the entomotoxigenecity of Cry proteins that accumulate as intracellular crystalline inclusion during sporulation. The purpose of this study was to produce alkaline-soluble ChiA74∆sp inclusions in B. thuringiensis, and to determine its effect on Cry crystal production, sporulation and toxicity to an important agronomical insect, Manduca sexta. To this end we deleted the secretion signal peptide-coding sequence of chiA74 (i.e. chiA74∆sp) and expressed it under its native promoter (pEHchiA74∆sp) or strong chimeric sporulation-dependent cytA-p/STAB-SD promoter (pEBchiA74∆sp) in Escherichia coli, acrystalliferous B. thuringiensis (4Q7) and B. thuringiensis HD1.


RESULTS
Based on mRNA analyses, up to ~9-fold increase in expression of chiA74∆sp was observed using the cytA-p/STAB-SD promoter. ChiA74∆sp (~70 kDa) formed intracellular inclusions that frequently accumulated at the poles of cells. ChiA74∆sp inclusions were dissolved in alkali and reducing conditions, similar to Cry crystals, and retained its activity in a wide range of pH (5 to 9), but showed a drastic reduction (~70%) at pH 10. Chitinase activity of E. coli-pEHchiA74∆sp was ~150 mU/mL, and in E. coli-pEBchiA74∆sp, 250 mU/mL. 4Q7-pEBchiA74∆sp and 4Q7-pEHchiA74∆sp had activities of ~127 mU/mL and ~41 mU/mL, respectively. The endochitinase activity in HD1-pEBchiA74∆sp increased 42x when compared to parental HD1 strain. HD1-pEBchiA74∆sp and HD1 harbored typical bipyramidal Cry inclusions, but crystals in the recombinant were ~30% smaller. Additionally, a 3x increase in the number of viable spores was observed in cultures of the recombinant strain when compared to HD1. Bioassays against first instar larvae of M. sexta with spore-crystals of HD1 or spore-crystal-ChiA74∆sp inclusions of HD1-pEBchiA74∆sp showed LC₅₀s of 67.30 ng/cm² and 41.45 ng/cm², respectively.


CONCLUSIONS
Alkali-labile ChiA74∆sp inclusion bodies can be synthesized in E. coli and B. thuringiensis strains. We demonstrated for the first time the applied utility of synthesis of ChiA74∆sp inclusions, Cry crystals and spores in the same sporangium of HD1, a strain used successfully worldwide to control economically significant lepidopteran pests of agriculture. Our findings will allow to us develop strategies to modify expression of ChiA74∆sp while maximizing Cry crystal synthesis in commercial strains of B. thuringiensis.
Universidad de Guanajuato Campus Irapuato-Salamanca, División Ciencias de la Vida, Posgrado en Biociencias, Irapuato, Guanajuato 36500, México. josebar@ugto.mx.
José Eleazar
JE
Barboza-Corona
Jorge Luis
JL
Delgadillo-Ángeles
José Cristóbal
JC
Castañeda-Ramírez
Uriel Eleazar
UE
Barboza-Pérez
Luz Edith
LE
Casados-Vázquez
Dennis K
DK
Bideshi
Ma Cristina
MC
del Rincón-Castro
D001413:Bacillus thuringiensis / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002688:Chitinases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D002479:Inclusion Bodies / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013171:Spores, Bacterial
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D002688:Chitinases