Fur-mediated regulation of hydrogen sulfide synthesis, stress response, and virulence in Edwardsiella piscicida.

The production of endogenous hydrogen sulfide (H2S) is an important phenotype of bacteria. H2S plays an important role in bacterial resistance to ROS and antibiotics, which significantly contributes to bacterial pathogenicity. Edwardsiella piscicida, the Gram-negative pathogen causing fish edwardsiellosis, has been documented to produce hydrogen sulfide. In the study, we revealed that Ferric uptake regulator (Fur) controlled H2S synthesis by activating the expression of phsABC operon. Besides, Fur participated in the bacterial defense against ROS and cationic antimicrobial peptides and modulated T3SS expression. Furthermore, the disruption of fur exhibited a significant in vivo colonization defect. Collectively, our study demonstrated the regulation of Fur in H2S synthesis, stress response, and virulence, providing a new perspective for better understanding the pathogenesis of Edwardsiella.

Gene Silencing Through CRISPR Interference in Bacteria: Current Advances and Future Prospects.

Functional genetic screening is an important method that has been widely used to explore the biological processes and functional annotation of genetic elements. CRISPR/Cas (Clustered regularly interspaced short palindromic repeat sequences/CRISPR-associated protein) is the newest tool in the geneticist's toolbox, allowing researchers to edit a genome with unprecedented ease, accuracy, and high-throughput. Most recently, CRISPR interference (CRISPRi) has been developed as an emerging technology that exploits the catalytically inactive Cas9 (dCas9) and single-guide RNA (sgRNA) to repress sequence-specific genes. In this review, we summarized the characteristics of the CRISPRi system, such as programmable, highly efficient, and specific. Moreover, we demonstrated its applications in functional genetic screening and highlighted its potential to dissect the underlying mechanism of pathogenesis. The recent development of the CRISPRi system will provide a high-throughput, practical, and efficient tool for the discovery of functionally important genes in bacteria.

Improvement of Curvulamine Production by Precursors Co-addition Strategy in Liquid Culture of Marine-Derived Fungus Curvularia sp. IFB-Z10.

Curvulamine, a novel scaffold alkaloid with remarkable selective antibacterial activity, is produced by marine fungus Curvularia sp. IFB-Z10. However, its deep pharmaceutical research and application are severely restricted by the low yield, which needs to be solved urgently. The purpose of this study was to improve curvulamine production via precursors co-addition strategy and further reveal the regulation mechanism. In this work, the optimal precursors co-addition conditions were firstly obtained, and curvulamine production achieved 166.74 mg/L with the supply of 250 mg/L alanine and 200 mg/L proline at 60 h, which was 4.08 times that of control. It was observed that under alanine and proline stimulation, fungus exhibited the morphology of a small-diameter compact pellet. Furthermore, the organic acid levels in central carbon metabolism (CCM) were declined with precursors supplement. Besides, precursors also induced the critical biosynthetic gene transcriptions. The above findings collectively promoted curvulamine synthesis. Finally, Curvularia sp. IFB-Z10 fermentation process was successfully established by feeding alanine and proline at 0.021 g/L/h and 0.017 g/L/h rate from 60 to 72 h, and curvulamine production reached 133.58 mg/L in a 5-L bioreactor. The information acquired would facilitate the enhancement of curvulamine yield in submerged fermentation and the research on synthesis regulation of other alkaloids.