The Role of the ribosome binding site sequence and spacer length between binding site and initiation codon on cry2 expression in Bacillus host

Abstract

The bacterium Bacillus thuringiensis (Bt) produces Cry toxins that possess toxic properties and can be used as biopesticides. Cry2Aa and Cry2Ac are among unusual subset of crystalline proteins possessing broad insect species specificity by exhibiting high specific activity against larvae from two insect orders, Lepidoptera and Diptera of agricultural and public health significance. The cry2Ac11 gene is located at third position (orf3) in operon comprising of three genes. It needs accessory proteins for crystal formation and high yield. Translation initiation is key rate-limiting step. It is well-known that stable structure at a ribosome binding site (RBS) impedes initiation. Modification in RBS-spacer region tunes translation initiation rates. Genetic manipulation of cry2Ac11 gene without helper protein was carried out in this study by optimizing ribosomal binding site and spacer region (RBS-ATG) in translation initiation region (TIR). The five different types of mutations were introduced in TIR to unveil inhibitory and excitatory effects on translation. These mutants are: 1), operSalI/RBSD and mut/RBSD in which downstream RBS (GGAGG) 6 bp downstream to native RBS was introduced in TIR of cry2Ac11 operon and gene; 2), mut/RBSF in which four nucleotides (ATGGG) were incorporated after RBS-ATG spacer region; 3), mut/RBSS in which overlapping start and two stop codons were introduced after RBS-ATG spacer region; 4), mut/RBSSP in which RBS-ATG spacer region was lengthened to 23 nucleotides; 5), mut/RBS2 in which consecutive two ATGs were incorporated in TIR. Secondary structures of mutants, estimated by CLC Main Workbench, revealed that mut/RBS2 RNA exhibits most stable structure in RBS-AUG region. RBS Calculator predicts high translation rate in mut/RBSD and mut/RBS2. Mutants were expressed in B. thuringiensis 4Q7 acrystalliferous strain. The transcriptomics-proteomics profiles of all cry2Ac11 constructs provide a unique opportunity to investigate how faithfully the transcriptional profile is manifested at the protein level. Therefore, in this study correlation between mRNA abundance and protein expression profiles in all Cry2Ac11 recombinant strains were also investigated. The highest transcript profile of B. thuringiensis 4Q7-mut/RBS2, (a mutant in which consecutive multiple AUG were introduced), was obtained by Real time PCR. Furthermore, SDS-PAGE profile of total cellular proteins indicated that overexpression of Cry2Ac11 (65kDa) was obtained in 4Q7-mut/RBS2. It was concluded that overexpression of Cry2Ac11 toxin without helper protein in mut/RBS2 mRNA was most likely due to presence of consecutive start codons (AUGs) in TIR. Presence of RBS in the single stranded part of moderately stable hairpin loop (ΔG = 8.7 kcal/mol) in mut/RBS2 facilitates the interaction of RBS to the complementary 16S rRNA sequences of 30S ribosomal subunit. In proposed model, multiple factors are thought to contribute in translation efficiency of mut/RBS2 (cry2Ac11 mutants without helper protein) which includes stabilizer sequence at 5′ and 3′ ends, the availability of the RBS for binding to the anti-SD of 16S rRNA of 30S ribosomal unit and optimal context of RBS-AUG region provided by multiple AUGs in TIR.