A Novel Antidipteran 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.

Pan-Genome Portrait of Bacillus mycoides Provides Insights into the Species Ecology and Evolution

“…Interestingly, in the “blue” cluster (with lower values of the a and π parameters) the average IS count was smaller than for the bacteria of the “turquoise” group (with higher values of the a and π parameters). Although the low number of bacilli studied does not allow any final conclusions to be drawn, these results suggest that among mobile genetic elements (MGEs), ISs play an important role in genetic variation of B. mycoides , as was also earlier observed for other members of the group, especially B. thuringiensis and B. cereus sensu stricto ( 25 , 45 ).…”

Conjugation across Bacillus cereus and kin: A review

“…While plasmids typically promote intercellular genetic moves, other MGEs, like insertion sequences (IS) and transposable elements (Tn), are usually responsible for intracellular mobility, i.e., moving from one location to another within the same genome. Transposable elements are for instance more prevalent in toxin plasmids of B. thuringiensis, namely about 30% of the total "thuringiensis" plasmid pool, as they often flank cry genes (Fiedoruk et al, 2017;Fayad et al, 2019Fayad et al, , 2021a. In natural environments, B. cereus s.l.…”

“…were characterized as conjugative, based either on experimental transfer evidence or on strong bioinformatics predictions (Table 1). Although recent works keep focusing on the importance (Bolotin et al, 2017;Gillis et al, 2018;Fayad et al, 2019Fayad et al, , 2021aZheng et al, 2020), few studies have investigated conjugative transfer per se. Furthermore, reported transfer events often consist in mobilization events without incriminating the implicated conjugative plasmid (Fguira et al, 2014).…”

IDOPS, a Profile HMM-Based Tool to Detect Pesticidal Sequences and Compare Their Genetic Context

“…Currently, none of them take into consideration the genetic environment of the genes coding for pesticidal proteins. Surrounding elements often include chaperones, crystallization domains, mobile elements, transporters, prophages, and virulence factors (Koni and Ellar, 1993 ; Shao et al, 2001 ; Elleuch et al, 2016 ; Adalat et al, 2017 ; Fayad et al, 2020 ; Lechuga et al, 2020 ). Moreover, the arrangement and distribution of such elements across genomes reveal crucial details about toxin functionality, host adaptation, diversification, and evolution of biopesticides (Khasdan et al, 2007 ; Peng et al, 2015 ; Ruffner et al, 2015 ; Fiedoruk et al, 2017 ; Zheng et al, 2017 ; Fayad et al, 2020 ; Wang et al, 2020 ).…”

“…Surrounding elements often include chaperones, crystallization domains, mobile elements, transporters, prophages, and virulence factors (Koni and Ellar, 1993 ; Shao et al, 2001 ; Elleuch et al, 2016 ; Adalat et al, 2017 ; Fayad et al, 2020 ; Lechuga et al, 2020 ). Moreover, the arrangement and distribution of such elements across genomes reveal crucial details about toxin functionality, host adaptation, diversification, and evolution of biopesticides (Khasdan et al, 2007 ; Peng et al, 2015 ; Ruffner et al, 2015 ; Fiedoruk et al, 2017 ; Zheng et al, 2017 ; Fayad et al, 2020 ; Wang et al, 2020 ). Hence, a more exhaustive approach would not only detect pesticidal sequences but also enable comparative genomics analysis of the candidate toxin in order to characterize the complete expression unit.…”