A novel anti-dipteran Bacillus thuringiensis strain: Unusual Cry toxin genes in a highly dynamic plasmid environment

Abstract

Bacillus thuringiensis emerged as a major bioinsecticide on the global market. It offers a valuable alternative to chemical products classically utilized to control pest insects. Despite the efficiency of several strains and products available on the market, the scientific community is always on the lookout for novel toxins that can replace or supplement the existing products. In this study, H3, a novel B. thuringiensis strain showing mosquitocidal activity, was isolated from Lebanese soil and characterized at an in vivo, genomic and proteomic levels. H3 parasporal crystal is toxic on its own but displays an unusual killing profile with a higher LC50 than the reference B. thuringiensis serovar israelensis crystal proteins. In addition, H3 has a different toxicity order: it is more toxic to Aedes albopictus and Anopheles gambiae than to Culex pipiens Whole genome sequencing and crystal analysis revealed that H3 can produce eleven novel Cry proteins, eight of which are assembled in genes with an orf1-gap-orf2 organization, where orf2 is a potential Cry4-type crystallization domain. Moreover, pH3-180, the toxin-carrying plasmid, holds a wide repertoire of mobile genetic elements that amount to ca 22% of its size., including novel insertion sequences and class II transposable elements Two other large plasmids present in H3 carry genetic determinants for the production of many interesting molecules - such as chitinase, cellulase and bacitracin - that may add up to H3 bioactive properties. This study therefore reports a novel mosquitocidal Bacillus thuringiensis strain with unusual Cry toxin genes in a rich mobile DNA environment.IMPORTANCE Bacillus thuringiensis, a soil entomopathogenic bacteria, is at the base of many sustainable eco-friendly bio-insecticides. Hence stems the need to continually characterize insecticidal toxins. H3 is an anti-dipteran B. thuringiensis strain, isolated from Lebanese soil, whose parasporal crystal contains eleven novel Cry toxins and no Cyt toxins. In addition to its individual activity, H3 showed potential as a co-formulant with classic commercialized B. thuringiensis products, to delay the emergence of resistance and to shorten the time required for killing. On a genomic level, H3 holds three large plasmids, one of which carries the toxin-coding genes, with four occurrences of the distinct orf1-gap-orf2 organization. Moreover, this plasmid is extremely rich in mobile genetic elements, unlike its two co-residents. This highlights the important underlying evolutionary traits between toxin-carrying plasmids and the adaptation of a B. thuringiensis strain to its environment and insect host spectrum.

FIG 1

Toxicity analysis of the spore-crystal mixture of B. thuringiensis strain H3 on third-instar larvae of A. albopictus, A. gambiae, and C. pipiens. Graphs show the percentage of survivors through time for a crystal-spore mixture concentration of 40 µg/ml.

FIG 2

Washed crystal protein profile on 12% SDS-PAGE of H3 and the reference B. thuringiensis sv. israelensis strain AM65-52. MW, molecular weight markers; sizes are indicated on the left.

FIG 3

SNP-based relationship dendrogram of several B. thuringiensis strains, with B. cytotoxicus NVH391-98 as an outgroup. Chromosomic DNA alignment and SNP extraction were done using progressiveMauve (37). The dendrogram was drawn using MEGA-X v10.0.5 (76).

FIG 4

Circular map of B. thuringiensis strain H3 toxin-carrying plasmid pH3-180. The block arrows in the outer circles indicate the predicted open reading frames (ORFs) in their direction of transcription, with or without functional annotation or relevant homologues. The black circle represents the GC content plotted using a sliding window, as the deviation from the average GC content of the entire sequence. The green/magenta circles represent the GC skew calculated, using a sliding window, as (G−C)/(G+C) and plotted as the deviation from the average GC skew of the entire sequence. ORFs encoding Cry toxins, transposable elements with their associated ORFs, and sporulation-related proteins are highlighted by blue, dark pink, and fuchsia block arrows, respectively. The composite transposons containing cry genes are indicated by gold arcs on the outermost circle.