Comparative genomics and quantitative proteomics reveal differentially produced proteins underlying virulence and host specificity in Bacillus thuringiensis

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Yury Malovichko1, Maria Belousova2, Elena Lukasheva3, Daria Gorbach4, Ekaterina Romanovskaya5, Christian Ihling6, Andrej Frolov7, Anton Nizhnikov8, Kirill Antonets9
1All-Russian Research Institute of Agricultural Microbiology, yu.malovichko@arriam.ru
2All-Russian Research Institute of Agricultural Microbiology, m.belousova@arriam.ru
3St. Petersburg State University, elena_lukasheva@mail.ru
4St. Petersburg State University, daria.gorba4@yandex.ru
5St. Petersburg State University, e.romanovskaya@spbu.ru
6Martin-Luther Universität Halle-Wittenberg, christian.ihling@pharmazie.uni-halle.de
7Martin-Luther Universität Halle-Wittenberg, Andrej.Frolov@ipb-halle.de
8All-Russian Research Institute of Agricultural Microbiology, a.nizhnikov@arriam.ru
9All-Russian Research Institute of Agricultural Microbiology, k.antonets@arriam.ru

Bacillus thuringiensis is a Gram-positive spore-forming bacterium known for its insecticidal activities. Although its features of high virulence and exceptional host specificity are wiely known and have conditioned its use as a source of novel biopesticides, molecular mechanisms underlying these traits remain elusive and are usually attributed to its repertoire of proteinaceous toxins. In this work we used combined proteogenomic approach to dissect <>. We used three different strains of B. thuringiensis belonging to the var. thuringiensis, darmstadtiensis and israeliensis and one derivate of B. thuringiensis var israeliensis, which lost the ability to produce Cry-toxins. By using hybrid Oxford Nanopore and Illumina sequencing we achieved replicon-level genome assemblies of the studied strains which, upon annotation, facilitated comparison of virulence factor repertoires and putative reasons of virulence loss in derivate strain, Further application of quantitative HPLC-Orbitrap-MS and proteome level-comparison between vegetative and sporulating cultures allowed us to identify the proteins, which were differentially produced in the strains at each stage. While proteins differentially produced at vegetative stage related mostly to cell metabolism with few virulence factors captured, those identified in spores included different spore coat proteins, flotillin-like proteins involved in in cell differentiation, exosporium proteins and a wide set of virulence factors including proteinaceous Cry toxins. Taking together the data obtained in this study indicate the impact of the accessory genes produced at various stages of colony development on pathogenicity-associated phenotypic traits in strains with different host ranges. To our knowledge, this is the first proteogenomic study of B. thuringiensis aimed to compare both genomic and proteomic profiles between serovars.

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