DNA secondary structure formation by DNA shuffling of the conserved domains of the Cry protein of Bacillus thuringiensis

Efrain H Pinzon,Alvaro M Florez,Sergio Orduz,Miguel O Suarez,Daniel A Sierra

doi:10.1186/s13628-017-0036-7

Efrain H Pinzon, Alvaro M Florez + Show 3 more

Open Access

https://doi.org/10.1186/s13628-017-0036-7

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Abstract

BackgroundThe Cry toxins, or δ-endotoxins, are a diverse group of proteins produced by Bacillus thuringiensis. While DNA secondary structures are biologically relevant, it is unknown if such structures are formed in regions encoding conserved domains of Cry toxins under shuffling conditions. We analyzed 5 holotypes that encode Cry toxins and that grouped into 4 clusters according to their phylogenetic closeness. The mean number of DNA secondary structures that formed and the mean Gibbs free energy left(overline{varDelta G}right) were determined by an in silico analysis using different experimental DNA shuffling scenarios. In terms of spontaneity, shuffling efficiency was directly proportional to the formation of secondary structures but inversely proportional to ∆G.ResultsThe results showed a shared thermodynamic pattern for each cluster and relationships among sequences that are phylogenetically close at the protein level. The regions of the cry11Aa, Ba and Bb genes that encode domain I showed more spontaneity and thus a greater tendency to form secondary structures (<∆G). In the region of domain III; this tendency was lower (>∆G) in the cry11Ba and Bb genes. Proteins that are phylogenetically closer to Cry11Ba and Cry11Bb, such as Cry2Aa and Cry18Aa, maintained the same thermodynamic pattern. More distant proteins, such as Cry1Aa, Cry1Ab, Cry30Aa and Cry30Ca, featured different thermodynamic patterns in their DNA.ConclusionThese results suggest the presence of thermodynamic variations associated to the formation of secondary structures and an evolutionary relationship with regions that encode highly conserved domains in Cry proteins. The findings of this study may have a role in the in silico design of cry gene assembly by DNA shuffling techniques.

Highlights

The Cry toxins, or δ-endotoxins, are a diverse group of proteins produced by Bacillus thuringiensis
A total of 162 f-ratio values were obtained as a product of the statistical analysis that summarized the thermodynamic behavior of cry gene clusters
Thermodynamic variations associated with the formation of DNA secondary structures under DNA shuffling conditions in a group of genes belonging to the cry family of B. thuringiensis was organized into 4 clusters according to their phylogenetic relationship

Summary

Introduction

The Cry toxins, or δ-endotoxins, are a diverse group of proteins produced by Bacillus thuringiensis. Among the in silico models available, some have focused on simulation and prediction [8, 9], while others have focused on the integration of kinetic elements in a Markov model [10] and on the optimization of the DNA shuffling reaction [11] These tools have used a Poissonexponential distribution [12] to simulate DNA fragmentation and have employed the unified calculation of free energy using the nearest-neighbor described by SantaLucia (1998) [13]. This calculation has been used as a thermodynamic parameter to predict the formation of secondary structures in computational tools such as UNAFold, Unified Nucleic Acid Folding [14] and NASP, Nucleic

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