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

Joaquín Gomis-Cebolla,Juan Ferré,Ana Scaramal Ricietto

doi:10.3390/toxins10050193

Joaquín Gomis-Cebolla, Juan Ferré + Show 1 more

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https://doi.org/10.3390/toxins10050193

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Journal: Toxins	Publication Date: May 10, 2018
Citations: 8	License type: CC BY 4.0

Affiliation: University of Valencia, Universidade Estadual de Londrina

Abstract

The combined analysis of genomic and proteomic data allowed us to determine which cry and vip genes are present in a Bacillus thuringiensis (Bt) isolate and which ones are being expressed. Nine Bt isolates were selected from Spanish collections of Bt based on their vip1 and vip2 gene content. As a first step, nine isolates were analyzed by PCR to select those Bt isolates that contained genes with the lowest similarity to already described vip1 and vip2 genes (isolates E-SE10.2 and O-V84.2). Two selected isolates were subjected to a combined genomic and proteomic analysis. The results showed that the Bt isolate E-SE10.2 codifies for two new vegetative proteins, Vip2Ac-like_1 and Sip1Aa-like_1, that do not show expression differences at 24 h vs. 48 h and are expressed in a low amount. The Bt isolate O-V84.2 codifies for three new vegetative proteins, Vip4Aa-like_1, Vip4Aa-like_2, and Vip2Ac-like_2, that are marginally expressed. The Vip4Aa-like_1 protein was two-fold more abundant at 24 h vs. 48 h, while the Vip4Aa-like_2 was detected only at 24 h. For Vip2Ac-like_2, no differences in expression were found at 24 h vs. 48 h. Moreover, the parasporal crystal of the E-SE10.2 isolate contains a single type of crystal protein, Cry23Aa-like, while the parasporal crystal from O-V84.2 contains three kinds of crystal proteins: 7.0–9.8% weight of Cry45Aa-like proteins, 35–37% weight of Cry32-like proteins and 2.8–4.3% weight of Cry73-like protein.

Highlights

Bacillus thuringiensis (Bt) is an entomopathogenic bacterium that produces several types of insecticidal proteins, such as Cry, Cyt, Vip, Sip, Mtx-like, and Bin-like proteins, along with other virulence factors contributing to its pathogenicity [1,2]
The Vip proteins are a family of proteins that are secreted during the vegetative growth phase and that have been classified into four groups according to their sequence homology: Vip1, Vip2, Vip3, and Vip4 [2]
A series of approaches have been used for isolating novel insecticidal protein genes from Bt, such as PCR, which has further evolved into specific applications to mining new insecticidal genes, such as PCR hybridization, PCR-RFLP, E-PCR and PCRSSCP [7,8,9,10,11]

Summary

Introduction

Bacillus thuringiensis (Bt) is an entomopathogenic bacterium that produces several types of insecticidal proteins, such as Cry, Cyt, Vip, Sip, Mtx-like, and Bin-like proteins, along with other virulence factors contributing to its pathogenicity [1,2]. Toxins 2018, 10, 193 toxins [3,4,5,6] In this arms race against insects, it is necessary to explore the potential of new insecticidal proteins for pest control. A series of approaches have been used for isolating novel insecticidal protein genes from Bt, such as PCR, which has further evolved into specific applications to mining new insecticidal genes, such as PCR hybridization, PCR-RFLP, E-PCR and PCRSSCP [7,8,9,10,11]. The construction of Bt DNA libraries, followed by screening by Western Blotting or a hybridization-based method, has been used to detect novel insecticidal protein genes [12,13,14]

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