A Strain of Bacillus thuringiensis Containing a Novel cry7Aa2 Gene that Is Toxic to Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)

Abstract

The genome of the Bacillus thuringiensis BM311.1 strain was sequenced and assembled in 359 contigs containing a total of 6,390,221 bp. The plasmidic ORF of a putative cry gene from this strain was identified as a potential novel Cry protein of 1138 amino acid residues with a 98% identity compared to Cry7Aa1 and a predicted molecular mass of 129.4 kDa. The primary structure of Cry7Aa2, which had eight conserved blocks and the classical structure of three domains, differed in 28 amino acid residues from that of Cry7Aa1. The cry7Aa2 gene was amplified by PCR and then expressed in the acrystalliferous strain BMB171. SDS-PAGE analysis confirmed the predicted molecular mass for the Cry7Aa2 protein and revealed that after in vitro trypsin incubation, the protein was degraded to a toxin of 62 kDa. However, when treated with digestive fluids from Leptinotarsa decemlineata larvae, one major proteinase-resistant fragment of slightly smaller size was produced. The spore and crystal mixture produced by the wild-type BM311.1 strain against L. decemlineata neonate larvae resulted in a LC50 value of 18.8 μg/mL, which was statistically similar to the estimated LC50 of 20.8 μg/mL for the recombinant BMB17-Cry7Aa2 strain. In addition, when this novel toxin was activated in vitro with commercial trypsin, the LC50 value was reduced 3.8-fold to LC50 = 4.9 μg/mL. The potential advantages of Cry7Aa2 protoxin compared to Cry7Aa1 protoxin when used in the control of insect pests are discussed.

Insecticidal Activity of Bacillus thuringiensis Proteins against Coleopteran Pests

“…Cry7Aa, formerly known as CryIIIC, is very toxic to Cylas species (Coleoptera: Brentidae) [ 56 ], even more than Cry3 protein, but it has no negative effects against Anthonomus grandis (Coleoptera: Curculionidae) or D. undecimpuntata [ 52 ]. Moreover, toxicity to Colorado potato beetle has been reported, but only after in vitro solubilization [ 52 ], which was countered by a recent report of a Cry7Aa-type protoxin which is active against L. decemlineata without any previous solubilization step [ 57 ]. Solubilized Cry7Ab is active against Henosepilachna vigintiomaculata (Coleoptera: Coccinellidae) and Acanthoscelides obtectus (Coleoptera: Chrysomelidae), but not against Anomala corpulenta (Coleoptera: Scarabaeidae) or Pyrrhalta aenescens (Coleoptera: Chrysomelidae) [ 57 , 58 ].…”

“…Moreover, toxicity to Colorado potato beetle has been reported, but only after in vitro solubilization [ 52 ], which was countered by a recent report of a Cry7Aa-type protoxin which is active against L. decemlineata without any previous solubilization step [ 57 ]. Solubilized Cry7Ab is active against Henosepilachna vigintiomaculata (Coleoptera: Coccinellidae) and Acanthoscelides obtectus (Coleoptera: Chrysomelidae), but not against Anomala corpulenta (Coleoptera: Scarabaeidae) or Pyrrhalta aenescens (Coleoptera: Chrysomelidae) [ 57 , 58 ]. Cry8-type proteins are toxic to a large number of coleopteran pests, particularly against species in the Scarabaeidae family [ 59 , 60 , 61 ].…”

Endophytic Bacillus spp. as a Prospective Biological Tool for Control of Viral Diseases and Non-vector Leptinotarsa decemlineata Say. in Solanum tuberosum L.

“…The entomopathogenic capacity of Bacillus thuringiensis strains to different orders of pests (Diptera, Lepidoptera, Hymenoptera, Coleoptera, Orthoptera, Hemiptera, etc.) involves synthesizing crystalline proteins that have insecticidal activity when ingested by a susceptible host ( Domínguez-Arrizabalaga et al, 2019 ), and now the ability of B. thuringiensis strains to invade plant internal tissues is of great interest, as well as insectotoxic properties of endophytic Bacillus subtilis strains.…”

Potential of Cry10Aa and Cyt2Ba, Two Minority δ-endotoxins Produced by Bacillus thuringiensis ser. israelensis, for the Control of Aedes aegypti Larvae

“…Bacillus electrocompetent cells were generated as described previously [ 52 ]. Briefly, bacteria were grown in 300 mL of Brain heart infusion broth (Pronadisa) at 28 °C under shaking conditions (200 rpm) until the culture reached an OD600 nm of 0.4.…”

“…Once pSTAB-cyt2Ba, pSTAB-cry10Aa and pSTAB-p19-cry11Aa-p20 were obtained, they were introduced into the acrystalliferous Bt strain BMB171. Bacillus electrocompetent cells were generated as described previously [52]. Briefly, bacteria were grown in 300 mL of Brain heart infusion broth (Pronadisa) at 28 • C under shaking conditions (200 rpm) until the culture reached an OD600 nm of 0.4.…”