Comparative Analysis of Genomics and Proteomics in the New Isolated Bacillus thuringiensis X022 Revealed the Metabolic Regulation Mechanism of Carbon Flux Following Cu2+ Treatment

Abstract

Bacillus thuringiensis (Bt) X022 is a novel strain isolated from soil in China, and showed strong insecticidal activity against several Lepidopteran pests. In this work, we performed whole genome sequencing of this Bt strain using the next-generation sequencing technology, and further conducted a comparative analysis with the proteomics data of the specific spore-release period based on LC-MS/MS approach. The Bt X022 genome consisted of one circular chromosomal DNA and seven plasmids, which were further functionally annotated using the RAST server. Comparative analysis of insecticidal substances showed that X022 contained genes coding for three Cry proteins (Cry1Ac, Cry1Ia and Cry2Ab) and a vegetative insecticidal protein (Vip3A). However, three insecticidal crystal proteins (ICPs) (Cry1Ca, Cry1Ac and Cry1Da) were detected by proteomics in the spore-release period. Moreover, a putative biosynthetic gene cluster and the metabolic pathway for poly-β-hydroxybutyrate in Bt X022 were deduced based on the comparative analysis of genomic and proteomic data, which revealed the metabolic regulation mechanism of carbon flux correlated with increased production of ICPs caused by Cu2+. Hence, these results provided a deeper understanding of the genetic background and protein expression profile of Bt X022. This study established a foundation for directed genetic modification and further application of this new isolated Bt strain.

Enhancing the insecticidal activity of new Bacillus thuringiensis X023 by copper ions

“…Cu 2+ can increase the expression level of insecticidal proteins of BtX023, and the enhanced toxicity to pests is similar to previous reports [16,20]. Previous reports indicated that Cu 2+ ions considerably affect the synthesis of PHB but does not significantly improve it.…”

“…Subsequently, the urea concentration in the sample was diluted to 1 M and subjected to enzymatic hydrolysis. These enzymatic fragments were separated and desalted by 2D-HPLC equipped with a strong cation-exchange column (BioBasic SCX; 0.32 mm × 100 mm, 5 μm) and a reversedphase column (BioBasic-C18; 0.1 mm × 150 mm, 5 μm) and then analyzed by LC-MS/MS using the Thermo Q-Exactive Plus (ThermoFisher, San Jose, CA, USA) equipped with an ultra-high performance liquid chromatography unit (Thermo Scientific Dionex Ultimate 3000) and a Nanospray Flex Ion-Source (Thermo Scientific) [16,20,[28][29][30]. After the original mass spectrometry data were converted into the mgf format file by the corresponding tools, the Maxquant software (according to the Bt protein library) was used to search for the identification and quantitative information extraction of the corresponding database.…”

A Genomic and Proteomic Approach to Identify and Quantify the Expressed Bacillus thuringiensis Proteins in the Supernatant and Parasporal Crystal

“…The quantity of the proteins expressed at the three different growth phases for the Bt isolates E-SE10.2 and O-V84.2 were 10.2–3.5% and 10.4–8.2%, 8.3–2.8%, respectively, of their genome encoded sequences. This low percentage of expressed proteins detected indicates that, in our experimental conditions, we only detect a small part of the predicted proteins by the genome data prediction, a phenomenon that has also been found in other studies [ 21 , 22 ]. The low percentage of detected expressed proteins should not be interpreted as that the rest of the proteins cannot be expressed, since they could do it under different growth conditions.…”

“…On the other hand, the development of the mass spectrometry (MS)-based proteomics has enabled the detection of proteins from complex mixtures from different stages of a microorganism [ 18 ]. The combination of the genomic and proteomic approaches is a very successful approach for validation and correction of predicted genomic coding information in a wide variety of organisms [ 18 , 19 , 20 , 21 , 22 ].…”

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

“…To date, both genomic and proteomic techniques are utilized in Bt studies, although the number of comparative proteomics papers [ 10 , 11 ] is considerably smaller than that of works on comparative genomics [ 12 , 13 , 14 , 15 ]. Ideally, the genomic and proteomic approaches should be used in parallel in order to detect virulence factors that are either missing in the proteome screening [ 16 ] or misannotated in the genomic data [ 17 ]. This combined approach is especially relevant when concerning the identification of Cry toxins, which tend to fall out of the scope in both proteomic [ 11 , 16 ] and genomic [ 18 ] studies.…”