A single point mutation in hmgA leads to melanin accumulation in Bacillus thuringiensis BMB181.

Bacillus thuringiensis BMB181 (Bt BMB181), with high melanin production, is an ideal candidate for industrial scale production of light-stable insecticides. However, its melanogenic pathways remain unclear. In the present study, we demonstrated that Bt BMB181 failed to produce melanin after treatment with mesotrione, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase in the homogentisic acid pathway of melanin synthesis. Heterologous expression experiments suggested that homogentisate-1,2-dioxygenase (HmgA) in Bt BMB171 functions normally, yet HmgA in Bt BMB181 had lost its activity, at least partly. Using the CRISPR-Cas9 system, the hmgA gene in Bt BMB171 was knocked out and the mutant strain gained the ability to produce melanin. Furthermore, the complemented strain reverted to the wild-type phenotype. In addition, overexpression of its own hmgA gene in Bt BMB181 also resulted in failure to produce the pigment. BLAST results indicated that the amino acid alteration (G272E) in HmgA of Bt BMB181 was caused by a single point mutation (815G→ A). The enzyme activity of purified HmgA171 was more than 10-fold higher than that of HmgA181. Finally, we determined that the mutation in hmgA was responsible for melanin accumulation in Bt BMB181. Our results provided new insights into the synthesis and regulation of melanin production in B.thuringiensis and will promote its future industrial application.

Scavenger receptor-C acts as a receptor for Bacillus thuringiensis vegetative insecticidal protein Vip3Aa and mediates the internalization of Vip3Aa via endocytosis.

The vegetative insecticidal proteins (Vip), secreted by many Bacillus thuringiensis strains during their vegetative growth stage, are genetically distinct from known insecticidal crystal proteins (ICPs) and represent the second-generation insecticidal toxins. Compared with ICPs, the insecticidal mechanisms of Vip toxins are poorly understood. In particular, there has been no report of a definite receptor of Vip toxins to date. In the present study, we identified the scavenger receptor class C like protein (Sf-SR-C) from the Spodoptera frugiperda (Sf9) cells membrane proteins that bind to the biotin labeled Vip3Aa, via the affinity magnetic bead method coupled with HPLC-MS/MS. We then certified Vip3Aa protoxin could interact with Sf-SR-C in vitro and ex vivo. In addition, downregulation of SR-C expression in Sf9 cells and Spodoptera exigua larvae midgut reduced the toxicity of Vip3Aa to them. Coincidently, heterologous expression of Sf-SR-C in transgenic Drosophila midgut significantly enhanced the virulence of Vip3Aa to the Drosophila larvae. Moreover, the complement control protein domain and MAM domain of Sf-SR-C are involved in the interaction with Vip3Aa protoxin. Furthermore, endocytosis of Vip3Aa mediated by Sf-SR-C correlates with its insecticidal activity. Our results confirmed for the first time that Sf-SR-C acts as a receptor for Vip3Aa protoxin and provides an insight into the mode of action of Vip3Aa that will significantly facilitate the study of its insecticidal mechanism and application.

NagRBt Is a Pleiotropic and Dual Transcriptional Regulator in Bacillus thuringiensis.

NagR, belonging to the GntR/HutC family, is a negative regulator that directly represses the nagP and nagAB genes, which are involved in GlcNAc transport and utilization in Bacillus subtilis. Our previous work confirmed that the chitinase B gene (chiB) of Bacillus thuringiensis strain Bti75 is also negatively controlled by YvoABt, the ortholog of NagR from B. subtilis. In this work, we investigated its regulatory network in Bti75 and found that YvoABt is an N-acetylglucosamine utilization regulator primarily involved in GlcNAc catabolism; therefore YvoABt is renamed as NagRBt. The RNA-seq data revealed that 27 genes were upregulated and 14 genes were downregulated in the ΔnagR mutant compared with the wild-type strain. The regulon (exponential phase) was characterized by RNA-seq, bioinformatics software, electrophoretic mobility shift assays, and quantitative real-time reverse transcription PCR. In the Bti75 genome, 19 genes that were directly regulated and 30 genes that were indirectly regulated by NagRBt were identified. We compiled in silico, in vitro, and in vivo evidence that NagRBt behaves as a repressor and activator to directly or indirectly influence major biological processes involved in amino sugar metabolism, nucleotide metabolism, fatty acid metabolism, phosphotransferase system, and the Embden-Meyerhof-Parnas pathway.

Complete genome sequence of Bacillus thuringiensis L-7601, a wild strain with high production of melanin.

Bacillus thuringiensis L-7601 (B. thuringiensis L-7601), belonging to Bacillus thuringiensis subsp. dendrolimus serotype H4a4b, is a wild-type strain which has the ability to produce melanin during the exponential phase of growth. The melanin produced is an excellent UV protective agent for the crystal insecticidal proteins. Here, we report the complete genome of B. thuringiensis L-7601 including one 5,790,408 bp chromosome and three plasmids. 6,519 CDSs and 150 RNA genes, including 106 tRNA genes, 39 rRNA genes and 5 ncRNA genes, were identified from the whole genome. In addition, our results indicated that homogentisic acid pathway is the melanogenic pathway in B. thuringiensis and accumulation of melanin is the consequence of hmgA frameshift mutant.