The Distribution of Several Genomic Virulence Determinants Does Not Corroborate the Established Serotyping Classification of Bacillus thuringiensis

Abstract

Bacillusthuringiensis, commonly referred to as Bt, is an object of the lasting interest of microbiologists due to its highly effective insecticidal properties, which make Bt a prominent source of biologicals. To categorize the exuberance of Bt strains discovered, serotyping assays are utilized in which flagellin serves as a primary seroreactive molecule. Despite its convenience, this approach is not indicative of Bt strains’ phenotypes, neither it reflects actual phylogenetic relationships within the species. In this respect, comparative genomic and proteomic techniques appear more informative, but their use in Bt strain classification remains limited. In the present work, we used a bottom-up proteomic approach based on fluorescent two-dimensional difference gel electrophoresis (2D-DIGE) coupled with liquid chromatography/tandem mass spectrometry(LC-MS/MS) protein identification to assess which stage of Bt culture, vegetative or spore, would be more informative for strain characterization. To this end, the proteomic differences for the israelensis-attributed strains were assessed to compare sporulating cultures of the virulent derivative to the avirulent one as well as to the vegetative stage virulent bacteria. Using the same approach, virulent spores of the israelensis strain were also compared to the spores of strains belonging to two other major Bt serovars, namely darmstadiensis and thuringiensis. The identified proteins were analyzed regarding the presence of the respective genes in the 104 Bt genome assemblies available at open access with serovar attributions specified. Of 21 proteins identified, 15 were found to be encoded in all the present assemblies at 67% identity threshold, including several virulence factors. Notable, individual phylogenies of these core genes conferred neither the serotyping nor the flagellin-based phylogeny but corroborated the reconstruction based on phylogenomics approaches in terms of tree topology similarity. In its turn, the distribution of accessory protein genes was not confined to the existing serovars. The obtained results indicate that neither gene presence nor the core gene sequence may serve as distinctive bases for the serovar attribution, undermining the notion that the serotyping system reflects strains’ phenotypic or genetic similarity. We also provide a set of loci, which fit in with the phylogenomics data plausibly and thus may serve for draft phylogeny estimation of the novel strains.

Draft Genome Sequencing of the Bacillus thuringiensis var. Thuringiensis Highly Insecticidal Strain 800/15

“…Nevertheless, there were noticeable inconsistencies even between the closest strains, such as S1307 and IS5056 or S908 and S601 (Figure 3d,e). This observation corroborates the fact that the distribution of determinants in Bt does not reflect taxonomic units and serological attributions [26]. Of all the homologs from VFDB, 98 were found in all the strains, and their abundance did not correlate with the number of loci coding for insecticidal moieties.…”

Recombine and succeed: a story of Cry toxins to expand the host range

“…Bt belongs to the B. cereus sensu lato along with infection agents, i.e., B. cereus sensu stricto and B. anthracis [15]. Nonetheless, commonly used genetic markers and complete genomes fail to distinguish these species; thus, Bt should be treated as a biovar that bears genes encoding specific insecticidal toxins [16][17][18]. The most prominent characteristic of Bt is the presence of crystalline inclusions termed the parasporal body that can account for up to 25% of the dry weight of the bacterium [4].…”

Bacillus thuringiensis (Bt) is more than a special agent for biological control of pests

“…16 Bt exert their toxicity by crystal proteins (δ--endotoxins) or other toxins synthesized during its growth and sporulation. 14,[17][18][19][20][21] Such proteins are very specific and safe to mammals' toxicity tests of Bt protein crystals for humans and animals support that the ingestion of these proteins does not allow them to solubilize, the Cry proteins rapidly degrade after being ingested. While both humans and animals lack specific recognition receptors for Cry proteins, necessary for them to be active.…”