Table_8_Bacillus subtilis BS-15 Effectively Improves Plantaricin Production and the Regulatory Biosynthesis in Lactiplantibacillus plantarum RX-8.XLSX

Plantaricin is a broad-spectrum bacteriocin produced by Lactiplantibacillus plantarum with significant food industry application potential. It was found that the plantaricin production of L. plantarum RX-8 was enhanced when co-culturing with Bacillus subtilis BS-15. This study, therefore, set out to explore how B. subtilis BS-15 induces biosynthesis of plantaricin. The effect of co-culturing with B. subtilis BS-15 on cell growth, plantaricin production, quorum-sensing (QS) signal molecule PlnA/autoinducer-2 (AI-2) secretion, as well as plantaricin biosynthesis gene cluster and AI-2 synthesis-associated gene expression, was investigated in bacteriocin-producer L. plantarum RX-8. When L. plantarum RX-8 and B. subtilis BS-15 were co-inoculated in Man–Rogosa–Sharp (MRS) for 20 h at an inoculum ratio of 1:1 (106:106 CFU/ml), the greatest plantaricin output (2,048 AU/ml) was obtained, rising by 32-fold compared with the monoculture of L. plantarum RX-8. Additionally, co-culture increased PlnA-inducing activity and AI-2 activity by 8- and 1.14-fold, respectively, over monoculture. RT-qPCR findings generated every 4 h (4–32 h) demonstrated that B. subtilis BS-15 remarkably improved the transcription of plnABCD and plnEF, and increased pfs and luxS transcription, even when using 200 mM D-ribose, a kind of AI-2 inhibitor. Based on the above findings, co-culturing with B. subtilis BS-15 as an environmental stimulus could activate the plantaricin induction via the PlnA-mediated intraspecies QS system and the AI-2-mediated interspecies QS system. Moreover, the inducing effect of PlnA and AI-2 in co-culture was independent. Differential proteomics analysis of B. subtilis BS-15 in co-culture indicated that bacteriocin-inducing regulatory mechanism may be related to flagellar assembly, peptidoglycan biosynthesis, anaerobic respiration, glycine cleavage system, or thiamin pyrophosphate biosynthesis.

植物乳杆菌素（Plantaricin）是由植物乳杆菌（Lactiplantibacillus plantarum）产生的广谱细菌素，在食品工业中具备显著的应用潜力。研究发现，当与枯草芽孢杆菌（Bacillus subtilis）BS-15共培养时，植物乳杆菌RX-8的植物乳杆菌素产量可得到显著提升。因此，本研究旨在探究枯草芽孢杆菌BS-15诱导植物乳杆菌素生物合成的具体机制。本研究以产细菌素的植物乳杆菌RX-8为研究对象，考察了与枯草芽孢杆菌BS-15共培养对其细胞生长、植物乳杆菌素产量、群体感应（quorum-sensing, QS）信号分子PlnA/自体诱导物-2（autoinducer-2, AI-2）分泌水平，以及植物乳杆菌素生物合成基因簇与AI-2合成相关基因转录表达水平的影响。当植物乳杆菌RX-8与枯草芽孢杆菌BS-15以1:1的接种比例（初始接种量均为10^6 CFU/ml）共同接种于MRS培养基（Man–Rogosa–Sharp）中培养20小时后，获得了最高的植物乳杆菌素效价（2048 AU/ml），相较于植物乳杆菌RX-8单独培养组提升了32倍。此外，共培养组的PlnA诱导活性与AI-2活性分别较单独培养组提升了8倍与1.14倍。每4小时一次（4~32小时）的实时荧光定量聚合酶链反应（RT-qPCR）检测结果显示，即便添加200 mM的D-核糖——一种AI-2抑制剂——，枯草芽孢杆菌BS-15仍可显著上调plnABCD与plnEF基因的转录水平，并提升pfs与luxS基因的转录活性。基于上述研究结果，与枯草芽孢杆菌BS-15共培养作为一种环境刺激因子，可通过PlnA介导的种内群体感应系统与AI-2介导的种间群体感应系统，激活植物乳杆菌素的合成诱导通路。此外，共培养体系中PlnA与AI-2的诱导作用相互独立。对共培养体系中的枯草芽孢杆菌BS-15开展差异蛋白质组学分析后发现，其介导细菌素产生的调控机制可能与鞭毛组装、肽聚糖生物合成、厌氧呼吸、甘氨酸裂解系统及焦磷酸硫胺素生物合成途径相关。