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Pine wood nematode disease is currently the most deadly forest disease in China, and the Monochamus alternatus is its primary vector. Controlling the M. alternatus is crucial for managing pine wood nematode disease. This study, based on the selected HasA (pGHKW4) secretory expression vector, used electroporation to combine the genetically modified high-toxicity toxin Cry3Aa-T with the entomopathogenic bacterium Yersinia entomophaga isolated from the gut of the M. alternatus. The SDS-PAGE and Western blotting techniques were employed to confirm the toxin protein's secretion capability. The engineered bacteria's genetic stability and effectiveness in controlling M. alternatus were assessed for their insecticidal activity. The results of the SDSPAGE and Western blotting analyses indicate that the HasA system effectively expresses toxin protein secretion, demonstrates certain genetic stability, and exhibits high insecticidal activity against M. alternatus. This study constructed a highly toxic entomopathogenic engineered bacterial strain against M. alternatus larvae, which holds significant implications for controlling M. alternatus, laying the foundation for subsequent research and application of this strain. [ABSTRACT FROM AUTHOR]

Abstract Background The Escherichia coli ER2566 strain (NC_CP014268.2) was developed as a BL21 (DE3) derivative strain and had been widely used in recombinant protein expression. However, like many other current RefSeq annotations, the annotation of the ER2566 strain was incomplete, with missing gene names and miscellaneous RNAs, as well as uncorrected annotations of some pseudogenes. Here, we performed a systematic reannotation of the ER2566 genome by combining multiple annotation tools with manual revision to provide a comprehensive understanding of the E. coli ER2566 strain, and used high-throughput sequencing to explore how the strain adapted under external pressure. Results The reannotation included noteworthy corrections to all protein-coding genes, led to the exclusion of 190 hypothetical genes or pseudogenes, and resulted in the addition of 237 coding sequences and 230 miscellaneous noncoding RNAs and 2 tRNAs. In addition, we further manually examined all 194 pseudogenes in the Ref-seq annotation and directly identified 123 (63%) as coding genes. We then used whole-genome sequencing and high-throughput RNA sequencing to assess mutational adaptations under consecutive subculture or overexpression burden. Whereas no mutations were detected in response to consecutive subculture, overexpression of the human papillomavirus 16 type capsid led to the identification of a mutation (position 1,094,824 within the 3′ non-coding region) positioned 19-bp away from the lacI gene in the transcribed RNA, which was not detected at the genomic level by Sanger sequencing. Conclusion The ER2566 strain was used by both the general scientific community and the biotechnology industry. Reannotation of the E. coli ER2566 strain not only improved the RefSeq data but uncovered a key site that might be involved in the transcription and translation of genes encoding the lactose operon repressor. We proposed that our pipeline might offer a universal method for the reannotation of other bacterial genomes with high speed and accuracy. This study might facilitate a better understanding of gene function for the ER2566 strain under external burden and provided more clues to engineer bacteria for biotechnological applications.

Pine wood nematode disease is currently the most deadly forest disease in China, and the Monochamus alternatus is its primary vector. Controlling the M. alternatus is crucial for managing pine wood nematode disease. This study, based on the selected HasA (pGHKW4) secretory expression vector, used electroporation to combine the genetically modified high-toxicity toxin Cry3Aa-T with the entomopathogenic bacterium Yersinia entomophaga isolated from the gut of the M. alternatus . The SDS-PAGE and Western blotting techniques were employed to confirm the toxin protein's secretion capability. The engineered bacteria's genetic stability and effectiveness in controlling M. alternatus were assessed for their insecticidal activity. The results of the SDS-PAGE and Western blotting analyses indicate that the HasA system effectively expresses toxin protein secretion, demonstrates certain genetic stability, and exhibits high insecticidal activity against M. alternatus. This study constructed a highly toxic entomopathogenic engineered bacterial strain against M. alternatus larvae, which holds significant implications for controlling M. alternatus , laying the foundation for subsequent research and application of this strain., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Han, Huang, Qi, Zhu, Hu, Wen, Long, Xu and Zhang.)

Background: The Escherichia coli ER2566 strain (NC_CP014268.2) was developed as a BL21 (DE3) derivative strain and had been widely used in recombinant protein expression. However, like many other current RefSeq annotations, the annotation of the ER2566 strain was incomplete, with missing gene names and miscellaneous RNAs, as well as uncorrected annotations of some pseudogenes. Here, we performed a systematic reannotation of the ER2566 genome by combining multiple annotation tools with manual revision to provide a comprehensive understanding of the E. coli ER2566 strain, and used high-throughput sequencing to explore how the strain adapted under external pressure. Results: The reannotation included noteworthy corrections to all protein-coding genes, led to the exclusion of 190 hypothetical genes or pseudogenes, and resulted in the addition of 237 coding sequences and 230 miscellaneous noncoding RNAs and 2 tRNAs. In addition, we further manually examined all 194 pseudogenes in the Ref-seq annotation and directly identified 123 (63%) as coding genes. We then used whole-genome sequencing and high-throughput RNA sequencing to assess mutational adaptations under consecutive subculture or overexpression burden. Whereas no mutations were detected in response to consecutive subculture, overexpression of the human papillomavirus 16 type capsid led to the identification of a mutation (position 1,094,824 within the 3′ non-coding region) positioned 19-bp away from the lacI gene in the transcribed RNA, which was not detected at the genomic level by Sanger sequencing. Conclusion: The ER2566 strain was used by both the general scientific community and the biotechnology industry. Reannotation of the E. coli ER2566 strain not only improved the RefSeq data but uncovered a key site that might be involved in the transcription and translation of genes encoding the lactose operon repressor. We proposed that our pipeline might offer a universal method for the reannotation of other bacterial genomes with high speed and accuracy. This study might facilitate a better understanding of gene function for the ER2566 strain under external burden and provided more clues to engineer bacteria for biotechnological applications. [ABSTRACT FROM AUTHOR]