Ruoxi Zhu,Liang Zhong,Yuying Xun,Shucheng Zheng,Yongtao Zhu,Wenlong Cai
Columnaris disease, caused by Flavobacterium columnare, represents one of the most economically devastating bacterial infections in global freshwater aquaculture. Despite its significant impact, the molecular mechanisms underlying F. columnare pathogenesis remain largely unexplored. Signal peptide peptidase A (SppA) plays a crucial role in bacterial protein secretion by degrading residual signal peptides after protein translocation, yet its function in F. columnare physiology and virulence has not been characterized. Here, we employed a targeted in-frame gene knockout approach to investigate the role of sppA in F. columnare. The ΔsppA mutant exhibited pleiotropic phenotypes including increased outer membrane vesicle (OMV) production (3.8-fold higher compared to the wild type), loss of gliding motility, enhanced efflux pump activity at the transcriptomic level, and reduced virulence. Transcriptomic profiling of the ΔsppA mutant revealed significant upregulation of genes involved in membrane stress response, including the genes in the MacAB-TolC efflux system, algU, and osmC, compared to the wild-type state. Importantly, survival assays demonstrated its virulence was significantly attenuated in freshwater Medaka (Oryzias latipes), with a 20% higher survival rate of fish compared to the wild type. Our findings reveal that SppA is essential for maintaining membrane homeostasis and normal cellular physiology in F. columnare and serves as one of the virulence factors during columnaris infection. These results provide important insights into the biological function of the sppA gene in F. columnare and highlight its role in bacterial protein secretion, membrane homeostasis, and pathogenesis.
p -Coumaric acid (para-hydroxycinnamic acid, p -HCA), as a kind of natural active substance from plant cell walls, is widely used in medicine, food, and cosmetics with its antioxidant, anticancer, and other properties. Biological production of p -HCA can be achieved via one-step deamination of the L -tyrosine by tyrosine ammonia-lyase (TAL). Here, a new highly active TAL ( Fc -TAL) from Flavobacterium columnare was identified via gene mining and heterologous expression . The purified Fc -TAL completely converts 22 mM L -tyrosine to p -HCA under alkaline condition (pH 9.5–10.0) at 55 °C. To increase enzymatic recyclability and reduce the bioproduction cost, Fc -TAL was immobilized on a silica nanospheres through silica-binding peptides (SiBP2) at pH 10. In comparison to free enzyme , the immobilized TAL SiBP2 exhibited higher stability, over 50% activity was retained even after 10 consecutive catalytic cycles. Further experiments revealed that an aliquot of immobilized enzyme (about 200 mg TAL) can transform 20 g L -tyrosine into17.8 g p -HCA with 96% yields and 98% purity through 5 consecutive reaction cycles. Our studies not only provided a strategy for enzyme immobilization in alkaline condition, but also developed a green process for p -coumaric acid biosynthesis with the highest conversion rate and yield reported to date.
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