# How Does _Bacillus thuringiensis_ Crystallize Such a Large Diversity of Toxins?
## 2 A Diverse Set of Mechanisms to Produce High Quantities of Toxins in \(\mathbf{Bt}\)  
The tremendous quantity of toxins produced by \(\mathbf{Bt}\), whose crystalline inclusion can represent up to 40% of the sporulated cells dry weight [9; 19], is essentially achieved by a fine and timely regulation of toxin gene transcription and by a stabilization of toxin transcripts [19; 20]. \(\mathbf{Bt}\) possesses the same transcriptional regulatory factors--called sigma factors (\(\sigma\))--that _Bacillus subtilis_ (\(\mathbf{Bs}\)) uses to regulate the expression of key genes at different stages of its sporulation [26]. Most genes encoding Cry and Cyt toxins are regulated by \(\sigma^{\text{E}}\) and \(\sigma^{\text{K}}\) that are active during sporulation [20; 22]. The promoter \(\sigma^{\text{H}}\), which is active during the transition from the vegetative growth to the sporulation phase, can also be found but it generally contributes little to the overall toxin gene expression [22]. Cry3 Aa is a notable exception as it uses \(\sigma^{\text{A}}\), active as early as the vegetative phase, for its production [19]. Each toxin gene exhibits a specific combination and arrangement of sigma factors in the 5\({}^{\prime}\) untranslated region (5\({}^{\prime}\) UTR). While sigma factors account for most of the \(\mathbf{Bt}\) toxins gene transcription regulation, other complementary mechanisms exist and have generally only been identified for specific \(\mathbf{Bt}\) strains and/or toxin genes so far [20]. As in all prokaryotes, the initiation of gene transcription in \(\mathbf{Bt}\) relies on ribosome binding sites (RBS) located between the promoter(s) and the'start' codon of the gene [27]. While the 'GGAGG' RBS motif is highly conserved in the _Bacillus_ genus [28], variations in the length and sequence of the translation initiation region (TIR), which encompasses the RBS region, can induce different secondary structures and strongly modulate the gene transcription level [29]. Two major mechanisms stabilizing the mRNA in its 5\({}^{\prime}\) and 3\({}^{\prime}\) ends have been identified in \(\mathbf{Bt}\), notably explaining why the half-life of \(\mathbf{Bt}\)_cry_ gene products (approx. 10 min) is particularly high [30]. In bacteria, mRNA decay is driven by different RNAses degrading mRNA from its 5\({}^{\prime}\) and/or 3\({}^{\prime}\) end [31]. Many toxin mRNAs are stabilized by repeated inverted sequences at their 3\({}^{\prime}\)-end, called stem-loops, forming secondary structures that block the mRNA degradation by 3\({}^{\prime}\)-exonucleases [32; 33]. Stabilization in 5\({}^{\prime}\) can also be performed thanks to a [PERSON] sequence present in 5\({}^{\prime}\) of the coding gene [34]. It is believed that this sequence stabilizes the mRNA by a \"ribosome stalling\" mechanism [35], promoting the recruitment of the 30S ribosomal subunit thanks to its sequence homology with the 3\({}^{\prime}\) end of 16S RNA [34]. Once bound, the subunit would protect the mRNA from degradation by 5\({}^{\prime}\) exonucleases, such as RNAse J, extending its half-life and allowing more toxin to be produced from a single mRNA [19; 32; 35]. Such a motif has initially been  
Figure 2: Schematic mode of action of \(\mathbf{Bt}\) toxins. Most \(\mathbf{Bt}\) toxins share a similar multistep mode of action [16]. They are produced as crystals during the sporulation of the bacterium. Spores and crystals are released in the aqueous environment where they are ingested by an insect larva (here, a mosquito larvae) The crystal then specifically dissolves due to the highly alkaline pH of the insect gut to release a protoxin. The propeptide located in the N- and/or C-terminal part of the protein (here, in red) is then cleaved off by digestive enzymes to release a protease-resistant mature toxin core (here, in green).