Ancona Veronica, Chatnaparat Tiyakhon, Zhao Youfu
Department of Crop Sciences, University of Illinois at Urbana-Champaign, 1201 W. Gregory Dr, Urbana, IL, 61801, USA.
Mol Genet Genomics. 2015 Aug;290(4):1265-76. doi: 10.1007/s00438-015-0988-8. Epub 2015 Jan 11.
In Erwinia amylovora, the Rcs phosphorelay system is essential for amylovoran production and virulence. To further understand the role of conserved aspartate residue (D56) in the phosphor receiver (PR) domain and lysine (K180) residue in the function domain of RcsB, amino acid substitutions of RcsB mutant alleles were generated by site-directed mutagenesis and complementation of various rcs mutants were performed. A D56E substitution of RcsB, which mimics the phosphorylation state of RcsB, complemented the rcsB mutant, resulting in increased amylovoran production and gene expression, reduced swarming motility, and restored pathogenicity. In contrast, D56N and K180A or K180Q substitutions of RcsB did not complement the rcsB mutant. Electrophoresis mobility shift assays showed that D56E, but not D56N, K180Q and K180A substitutions of RcsB bound to promoters of amsG and flhD, indicating that both D56 and K180 are required for DNA binding. Interestingly, the RcsBD56E allele could also complement rcsAB, rcsBC and rcsABCD mutants with restored virulence and increased amylovoran production, indicating that RcsB phosphorylation is essential for virulence of E. amylovora. In addition, mutations of T904 and A905, but not phosphorylation mimic mutation of D876 in the PR domain of RcsC, constitutively activate the Rcs system, suggesting that phosphor transfer is required for activating the Rcs system and indicating both A905 and T904 are required for the phosphatase activity of RcsC. Our results demonstrated that RcsB phosphorylation and dephosphorylation, phosphor transfer from RcsC are essential for the function of the Rcs system, and also suggested that constitutive activation of the Rcs system could reduce the fitness of E. amylovora.
在梨火疫病菌中，Rcs磷信号转导系统对于支链淀粉产生和毒性至关重要。为了进一步了解保守天冬氨酸残基（D56）在磷受体（PR）结构域以及赖氨酸（K180）残基在RcsB功能结构域中的作用，通过定点诱变产生了RcsB突变等位基因的氨基酸替换，并对各种rcs突变体进行了互补实验。RcsB的D56E替换模拟了RcsB的磷酸化状态，对rcsB突变体起到了互补作用，导致支链淀粉产量增加、基因表达增强、群体游动性降低，并恢复了致病性。相比之下，RcsB的D56N以及K180A或K180Q替换未能对rcsB突变体起到互补作用。电泳迁移率变动分析表明，RcsB的D56E替换而非D56N、K180Q和K180A替换能够与amsG和flhD的启动子结合，这表明D56和K180对于DNA结合都是必需的。有趣的是，RcsBD56E等位基因也能够互补rcsAB、rcsBC和rcsABCD突变体，恢复其毒性并增加支链淀粉产量，这表明RcsB磷酸化对于梨火疫病菌的毒性至关重要。此外，RcsC的PR结构域中T904和A905的突变而非D876的磷酸化模拟突变能够组成型激活Rcs系统，这表明磷转移对于激活Rcs系统是必需的，同时也表明A905和T904对于RcsC的磷酸酶活性都是必需的。我们的结果表明，RcsB的磷酸化和去磷酸化、从RcsC进行的磷转移对于Rcs系统的功能至关重要，同时也表明Rcs系统的组成型激活可能会降低梨火疫病菌的适应性。
