Bacillus thuringiensis differs from the closely related Bacillus cereus group species by its ability to produce crystalline inclusions. The production of these crystals mainly results from the expression of the cry genes, from the stability of their transcripts and from the synthesis, accumulation and crystallization of large amounts of insecticidal Cry proteins. This process normally coincides with sporulation and is regulated by various factors operating at the transcriptional, post-transcriptional, metabolic and post-translational levels.

Bacillus thuringiensis GabR is a Sigma 54-dependent transcriptional activator containing three typical domains, an N-terminal regulatory domain Per-ARNT-Sim (PAS), a central AAA+ (ATPases associated with different cellular activities) domain and a C-terminal helix-turn-helix (HTH) DNA binding domain. GabR positively regulates the expression of the gabT gene of the gab gene cluster, which is responsible for the γ-aminobutyric acid (GABA) shunt. Purified GabR was shown to specifically bind to a repeat region that mapped 58 bp upstream of the gabT start codon. The specific signal factors GABA and succinic semialdehyde (SSA) activated gabT expression, whereas GABA- and SSA-inducible gabT transcription was abolished in sigL and gabR mutants. GABA and SSA did not induce the expression of either SigL or GabR. Deletion of the PAS domain of GabR resulted in increased gabT transcriptional activity, both in the presence and absence of GABA. This study identified the GabR-binding site on the gabT promoter; however, GabR does not bind to its own promoter. gabT transcription is induced by GABA and SSA, and inducible expression is dependent on SigL and activated by GabR. The PAS domain in GabR is repressing its enhancer transcriptional activity on the gabT promoter. Repression is released upon GABA addition, whereupon transcription is induced.

The cell death and survival paradox in various biological processes requires clarification. While spore development causes maternal cell death in Bacillus species, the involvement of other cell death pathways in sporulation remains unknown. Here, we identified a novel ArsR family transcriptional regulator, CdsR, and found that the deletion of its encoding gene cdsR causes cell lysis and inhibits sporulation. To our knowledge, this is the first report of an ArsR family transcriptional regulator governing cell death. We found that CdsR directly repressed lrgAB expression. Furthermore, lrgAB overexpression resulted in cell lysis without sporulation, akin to the cdsR mutant, suggesting that LrgAB, a holin-like protein, induces cell death in Bacillus spp. The lrgAB mutation increases abnormal cell numbers during spore development. In conclusion, we propose that a novel repressor is vital for inhibiting LrgAB-dependent cell lysis.

Bacillus thuringiensis produces insecticidal crystal proteins encoded by cry or cyt genes and targets a variety of insect pests. We previously found that a strong promoter of a DeoR family transcriptional regulator (HD73_5014) can efficiently drive cry1Ac expression in B. thuringiensis HD73. Here, we investigated the regulation of neighbor genes by HD73_5014. The HD73_5014 homologs are widely distributed in Gram-positive bacterial species. Its neighbor genes include pepV, rsuA, and ytgP, which encode dipeptidase, rRNA pseudouridine synthase and polysaccharide biosynthesis protein, respectively. The four open reading frames (ORFs) are organized to be a pepR gene cluster in HD73. RT-PCR analysis revealed that the rsuA and ytgP genes formed a transcriptional unit (rsuA-ytgP operon), while pepV formed a transcriptional unit in HD73. Promoter-lacZ fusion assays showed that the pepV and rsuA-ytgP promoters are regulated by HD73_5014. EMSA experiments showed that HD73_5014 directly binds to the pepV promoter region but not to the rusA-ytgP promoter region. Thus, the HD73_5014 transcriptional regulator, which controls the expression of the dipeptidase pepV, was named PepR (dipeptidase regulator). We also confirmed the direct regulation between PepR and PepV by the increased sensitivity to vancomycin in ΔpepV and ΔpepR mutants compared to HD73.

Cellobiose, a β-1,4-linked glucose dimer, is a major cellodextrin resulting from the enzymatic hydrolysis of cellulose. It is a major source of carbon for soil bacteria. In bacteria, the phosphoenolpyruvate (PEP): carbohydrate phosphotransferase system (PTS), encoded by the cel operon, is responsible for the transport and utilization of cellobiose. In this study, we analyzed the transcription and regulation of the cel operon in Bacillus thuringiensis (Bt). The cel operon is composed of five genes forming one transcription unit. β-Galactosidase assays revealed that cel operon transcription is induced by cellobiose, controlled by Sigma54, and positively regulated by CelR. The HTH-AAA⁺ domain of CelR recognized and specifically bound to three possible binding sites in the celA promoter region. CelR contains two PTS regulation domains (PRD1 and PRD2), which are separated by two PTS-like domains-the mannose transporter enzyme IIA component domain (EIIA^Man) and the galactitol transporter enzyme IIB component domain (EIIB^Gat). Mutations of His-546 on the EIIA^Man domain and Cys-682 on the EIIB^Gat domain resulted in decreased transcription of the cel operon, and mutations of His-839 on PRD2 increased transcription of the cel operon. Glucose repressed the transcription of the cel operon and catabolite control protein A (CcpA) positively regulated this process by binding the cel promoter. In the celABCDE and celR mutants, PTS activities were decreased, and cellobiose utilization was abolished, suggesting that the cel operon is essential for cellobiose utilization. Bt has been widely used as a biological pesticide. The metabolic properties of Bt are critical for fermentation. Nutrient utilization is also essential for the environmental adaptation of Bt. Glucose is the preferred energy source for many bacteria, and the presence of the phosphotransferase system allows bacteria to utilize other sugars in addition to glucose. Cellobiose utilization pathways have been of particular interest owing to their potential for developing alternative energy sources for bacteria. The data presented in this study improve our understanding of the transcription patterns of cel gene clusters. This will further help us to better understand how cellobiose is utilized for bacterial growth.