Zhang Qingxia, Xing Chenglin, Kong Xiangwei, Wang Cheng, Chen Xijun
College of Horticulture and Plant Protection, Yangzhou University, Yangzhou, China.
Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, China.
Front Microbiol. 2021 May 14;12:667637. doi: 10.3389/fmicb.2021.667637. eCollection 2021.
Many strains produce the antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (2,4-DAPG), both of which have antimicrobial properties. The biosynthesis of these metabolites is typically controlled by multiple regulatory factors. Virulence factor regulator (Vfr) is a multifunctional DNA-binding regulator that modulates 2,4-DAPG biosynthesis in FD6. However, the mechanism by which Vfr regulates this process remains unclear. In the present study, chromatin immunoprecipitation of FLAG-tagged Vfr and nucleotide sequencing analysis were used to identify 847 putative Vfr binding sites in FD6. The consensus Vfr binding site predicted from nucleotide sequence alignment is TCACA. The qPCR data showed that Vfr positively regulates the expression of and , and the expression of these genes was characterized in detail. The purified recombinant Vfr bound to an approximately 240-bp fragment within the and upstream regions that harbor putative Vfr consensus sequences. Using electrophoretic mobility shift assays, we localized Vfr binding to a 25-bp fragment that contains part of the Vfr binding region. Vfr binding was eliminated by mutating the TACG and CACA sequences in and , respectively. Taken together, our results show that Vfr directly regulates the expression of the 2,4-DAPG operon by binding to the upstream regions of both the and genes. However, unlike other Vfr-targeted genes, Vfr binding to FD6 does not require an intact binding consensus motif. Furthermore, we demonstrated that expression is autoregulated in this bacterium. These results provide novel insights into the regulatory role of Vfr in the biocontrol agent .
许多菌株会产生抗生素绿脓菌素（PLT）和2,4 - 二乙酰基间苯三酚（2,4 - DAPG），这两种物质都具有抗菌特性。这些代谢产物的生物合成通常受多种调控因子控制。毒力因子调节蛋白（Vfr）是一种多功能DNA结合调节蛋白，可调节FD6中2,4 - DAPG的生物合成。然而，Vfr调节这一过程的机制仍不清楚。在本研究中，利用带有FLAG标签的Vfr进行染色质免疫沉淀和核苷酸测序分析，以鉴定FD6中的847个假定Vfr结合位点。通过核苷酸序列比对预测的Vfr共有结合位点是TCACA。qPCR数据表明，Vfr正向调节 和 的表达，并对这些基因的表达进行了详细表征。纯化的重组Vfr与 和 上游区域内一个含有假定Vfr共有序列的约240 bp片段结合。使用电泳迁移率变动分析，我们将Vfr结合定位到一个包含部分Vfr结合区域的25 bp片段上。分别突变 和 中的TACG和CACA序列可消除Vfr结合。综上所述，我们的结果表明，Vfr通过结合 和 基因的上游区域直接调节2,4 - DAPG操纵子的表达。然而，与其他Vfr靶向基因不同，Vfr与FD6的结合不需要完整的结合共有基序。此外，我们证明了该细菌中 的表达是自我调节的。这些结果为Vfr在生物防治剂 中的调节作用提供了新的见解。
