Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
PLoS One. 2012;7(9):e45038. doi: 10.1371/journal.pone.0045038. Epub 2012 Sep 18.
In this study, we attempted to understand the role of an orphan gene amyR in Erwinia amylovora, a functionally conserved ortholog of ybjN in Escherichia coli, which has recently been characterized. Amylovoran, a high molecular weight acidic heteropolymer exopolysaccharide, is a virulent factor of E. amylovora. As reported earlier, amylovoran production in an amyR knockout mutant was about eight-fold higher than that in the wild type (WT) strain of E. amylovora. When a multicopy plasmid containing the amyR gene was introduced into the amyR mutant or WT strains, amylovoran production was strongly inhibited. Furthermore, amylovoran production was also suppressed in various amylovoran-over-producing mutants, such as grrSA containing multicopies of the amyR gene. Consistent with amylovoran production, an inverse correlation was observed between in vitro expression of amyR and that of amylovoran biosynthetic genes. However, both the amyR knockout mutant and over-expression strains showed reduced levan production, another exopolysaccharide produced by E. amylovora. Virulence assays demonstrated that while the amyR mutant was capable of inducing slightly greater disease severity than that of the WT strain, strains over-expressing the amyR gene did not incite disease on apple shoots or leaves, and only caused reduced disease on immature pear fruits. Microarray studies revealed that amylovoran biosynthesis and related membrane protein-encoding genes were highly expressed in the amyR mutant, but down-regulated in the amyR over-expression strains in vitro. Down-regulation of amylovoran biosynthesis genes in the amyR over-expression strain partially explained why over-expression of amyR led to non-pathogenic or reduced virulence in vivo. These results suggest that AmyR plays an important role in regulating exopolysaccharide production, and thus virulence in E. amylovora.
在这项研究中，我们试图了解孤儿基因 amyR 在韧皮部坏死欧文氏菌（Erwinia amylovora）中的作用。该基因在大肠杆菌中具有功能保守的 ybjN 直系同源物，最近已被鉴定。果聚糖是一种高分子量酸性杂多糖胞外多糖，是韧皮部坏死欧文氏菌的毒力因子。正如之前报道的那样，amyR 敲除突变体中的果聚糖产量比野生型（WT）菌株高约 8 倍。当将含有 amyR 基因的多拷贝质粒导入 amyR 突变体或 WT 菌株时，果聚糖的产量受到强烈抑制。此外，在各种果聚糖过度产生的突变体中，如含有 amyR 基因多拷贝的 grrSA，果聚糖的产生也受到抑制。与果聚糖的产生一致，amyR 的体外表达与果聚糖生物合成基因的表达呈负相关。然而，amyR 敲除突变体和过表达菌株的甘露聚糖产量均降低，甘露聚糖是韧皮部坏死欧文氏菌产生的另一种胞外多糖。毒力测定表明，虽然 amyR 突变体能够引起比 WT 菌株略高的疾病严重程度，但过表达 amyR 的菌株不会在苹果嫩枝或叶片上引起疾病，仅在不成熟的梨果实上引起疾病减轻。微阵列研究表明，在 amyR 突变体中，果聚糖生物合成和相关膜蛋白编码基因高度表达，但在体外 amyR 过表达菌株中下调。amyR 过表达菌株中果聚糖生物合成基因的下调部分解释了为什么 amyR 的过表达导致体内非致病性或毒力降低。这些结果表明，AmyR 在调节胞外多糖的产生，从而调节韧皮部坏死欧文氏菌的毒力方面发挥着重要作用。
