Bt Research 2025, Vol.16, No.5, 194-203 http://microbescipublisher.com/index.php/bt 198 typical example is that Bt strains change virulence spectrum and regulate network architecture by losing or obtaining plasmids. When Bt obtains a new toxin plasmid, the toxin gene on the plasmid needs to be integrated into the regulatory system of the host chromosome, otherwise it may not be effectively expressed. Research has found that some Bt plasmids also encode their own regulatory proteins, which can specifically activate the expression of toxin genes of the plasmid, making them not completely dependent on the σ factor of the host chromosome (Xiang and Wu, 2024). In the laboratory, artificial mutations can also reshape the Bt network. For example, through physical and chemical complex mutagenesis, continuous directed evolution screening of Bt strains can be obtained to obtain mutant strains with increased virulence. These mutant strains tend to change in regulatory elements, such as promoter point mutations leading to higher transcriptional efficiency, or negative regulatory gene inactivation leading to de-suppression of toxin genes (Qin et al., 2021). 5 Regulatory Mechanism of Bt ToxinGene 5.1 Promoter activity and regulatory factors of Cry gene The cry gene is one of the focus of research on Bt gene regulation network. The promoter structure and activity of different cry genes differ, giving them different expression patterns during the development cycle of Bt cells. Taking the cry1 gene example, the study found that its promoter region usually contains conserved sequences that bind to σ^E and σ^K factors, and is therefore activated to transcription mainly at the sporoblast stage. At the same time, there is often an A+T enrichment region upstream of the promoter, which can be bound by regulatory proteins such as Spo0A and AbrB, thereby limiting the expression of cry1 to appropriate times. The TcdR was discovered early on, which was the sigma factor protein that activates the cyt toxin gene in the Israeli subspecies Bt, which specifically initiates the transcription of the Cyt gene; and there is recent evidence that some transposal regulatory proteins may also affect the transcription initiation intensity of adjacent toxin genes. The methylation state of the promoter also regulates cry gene expression. If a promoter region is modified by DNA methyltransferase, it may hinder RNA polymerase binding, resulting in a decrease in the expression of the toxin gene. In terms of regulatory factors, in addition to global Spo0A, σ^E, etc., some local regulators play an important role in cry gene expression. Although PlcR does not directly regulate crying, its downstream products such as certain proteases can degrade immature procrystals and indirectly affect effective toxin formation (Zhang et al., 2018). 5.2 Case analysis: transcriptional regulatory characteristics of high virulence Bt strains Highly virulent Bt strains usually have stronger insecticidal protein expression and higher pathogenic factor activity, which is often attributed to their special regulatory network status. Taking the Bt Israeli subspecies (Bti), as an example, this strain is known for its high toxicity to Dipteran larvae. Proteome studies show that Bti not only produces major toxin proteins such as Cry4 and Cry11, but also expresses a variety of concellular crystal proteins and secondary metabolites, forming a "virulence factor cocktail." From a regulatory perspective, the Bti genome carries more copies and plasmids of toxin genes than the general Bt, which means that its regulatory network must coordinate the efficient expression of multiple toxin genes at the same time (Wirth et al., 2003). Transcriptome analysis showed that the mRNA abundance of toxin genes in the spore formation process of the highly virulent strain Bt 4.0718 was significantly higher than that of the model laboratory strain, and the peak expression time was slightly earlier. This may be due to the high expression of certain regulatory factors in strain 4.0718 or the mutation of enhanced activity. 5.3 Comparative study with low virulence strains Comparing highly virulent Bt strains with low virulent or non-virulent Bt strains is an effective way to elucidate the regulatory mechanism of toxin genes. Low virulent strains provide a "lost background" that can help identify regulatory factors specific to high virulent strains. Comparative studies show that low-virulence strains often have the following differences: First, the number of toxin genes is small or completely missing, resulting in no toxins that can be expressed even if the regulatory system is intact. More importantly, its regulatory network may be more inclined toward survival rather than pathogenic directions (Wang et al., 2025). Second, there may be variations in the global regulatory factors of low-virulence strains. For example, some low-virulent strains may
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