# How Does _Bacillus thuringiensis_ Crystallize Such a Large Diversity of Toxins?
## 1 Introduction  
_Bacillus thuringiensis_ (\(Bt\)) is a natural crystal maker that has evolved over millions of years to form a set of hundreds of subspecies, each crystallizing one or more toxins required for infecting insect larvae [1]. \(Bt\) is able to produce a high diversity of toxins belonging to nine structurally different categories, of which six are produced as crystalline inclusions during \(Bt\) sporulation (Figure 1) [2, 3, 4]. Each \(Bt\) subspecies is specific to an invertebrate group, which is determined by the particular cocktail of crystalline toxin(s) that it expresses [5]. This explains why it is the most used biological insecticide worldwide to control different insect species considered as agricultural pests (e.g., caterpillars or mealworms) or as threats for human health (e.g., mosquitoes) [6]. Despite strong structural differences (Figure 1), all \(Bt\) toxins have a similar mode of action and their crystals exhibit shared properties (Figure 2). Indeed, the purpose of toxin crystallization is for \(Bt\) to ensure the long-term storage of its main virulence factors in an aqueous environment so that they remain functional until they are ingested together with the spores by the insect host. The release of toxins is controlled by the specific dissolution of the crystals at high pHs only (\(>\)10), which correspond to the insect intestinal pH and further participate to \(Bt\) host specificity. Altogether, this shows that \(Bt\) has developed a wide array of strategies to achieve similar crystal formation and dissolution properties for proteins of drasticallydifferent sequences and structures. Considering that the biology and ecology of \(Bt\) has been extensively studied for decades and that several molecular tools have been developed to enable the recombinant production of proteins in \(Bt\)[7; 8; 9], one could wonder whether the crystallization machinery of \(Bt\) can be hijacked to produce crystalline formulations of proteins of interest for structural biology and biotechnological applications. This two-articles series aims at addressing the following question: can \(Bt\) be turned into a custom crystal biofactory and how? A prerequisite for using \(Bt\) to produce custom microcrystals is to characterize the mechanisms allowing \(Bt\) to crystallize such a wide variety of toxins. This topic has already been addressed several times in excellent comprehensive review articles, which we invite the reader to explore for more detailed information on specific aspects [18; 19; 20; 21; 22; 23]. In this review, we centralize the most relevant information in regard with the general question of this series by adopting a comparative approach between in vitro and in vivo crystallization processes to highlight how \(Bt\) can control the specific crystallization of a large set of diverse toxins while sustaining the production of many membrane and soluble proteins involved in spore formation [24; 25]. Most notably, we develop the diversity of the mechanisms used to produce a high quantity of toxins (Section 2), the intrinsic crystalline characteristics of toxins (Section 3), the role of accessory proteins (Section 4) and eventually how a fine equilibrium between these three factors conditions toxins crystallization in \(Bt\) (Section 5). Then, we conclude by exploring the past, current and future contribution of structural biology accompanying its (r)evolution (Section 6). The aim of this article is thus to provide the necessary knowledge on the \(Bt\) system for a large range of multidisciplinary readers to understand the relevance and feasibility of the main question addressed. Figure 1: \(Bt\) can crystallize toxins belonging to six structurally different groups. The structure of a representative toxin is shown for each group: Cry1 Ac [10], Cyt1 Aa [11], Gpp34 Aa (Cry34 Ab) [12], Tpp35 Ab (Cry35 Ab) [12], Mpp51 Aa (Cry51 Aa) [13], and App6 Aa (Cry6 Aa) [14]. Each group exhibits structural peculiarities.