大腸桿菌DUF28蛋白質在氧化逆境反應之功能研究

Abstract

除了在細菌之外， DUF28 蛋白質家族廣泛存在各物種，但對其功能所知甚少，僅知此蛋白在 Pseudomonas aeruginosa 中參與 swarming 泳動性與調節菌量感應反應 (quorum sensing)，在哺乳類動物中則扮演 COX I 的轉譯活化子 (translation activator) 的角色。本研究發現部分細菌具有兩個 DUF28 蛋白質，十分特別，故針對大腸桿菌之兩個 DUF28 蛋白 YeeN 及 YebC 進行功能分析，尤著重在氧化逆境反應之功能。序列發現在不同菌種的胺基酸序列相似度不盡相同，推測此蛋白質保有相似的立體結構。突變株特性分析結果顯示 ΔyeeN 及 ΔyebC 之泳動能力都有明顯增加，但唯有 ΔyeeN 抵抗 paraquat 氧化逆境的耐受度有明顯增加，互補實驗亦進一步證實這些結果；此外， YeeN 量的累積與逆境因子易造成此蛋白質在細胞質中呈點狀分布，而 YebC 除在生長死亡期會有段狀分佈的情況外，大多均勻分佈；啟動子分析結果顯示， yeeN 具有自己的啟動子且會受氧化逆境的抑制，而 yebC 啟動子則在細胞生長後期才具活性且不會受到氧化逆境的影響。進一步分析可能調節 yeeN 表現量之上游基因發現， recA 在氧化逆境下可調節 yeeN ； relA 在正常情況下可正向調控 yeeN ，但並未直接參與 yeeN 在氧化逆境條件之調控； hns 於生長後期會抑制 yeeN ，並可能參與 yeeN 負調控鞭毛合成相關基因之功能。綜合以上結果可知，大腸桿菌中 YeeN 與 YebC 皆可調節細菌之泳動力，但目前確認 YeeN 也同時參與氧化逆境反應，本研究首次報導兩個同為 DUF28 蛋白質家族成員之基因，彼此功能有同也有異。

DUF28 protein family ubiquitously exists in all organisms, except Archaea; however, information on their function is very limited. Previous studies demonstrated that DUF28 is involved in Pseudomonas aeruginosa swarming motility and quorum sensing, and functions as a translational activator of COX I in mammals. Interestingly, this study revealed the existence of two DUF28 homologs in some eubacteria. The aim of this study was to investigate functions of the two E. coli DUF28 proteins, namely YeeN and YebC, particularly their function in oxidative stress responses. Sequence comparisons revealed that bacterial DUF28 proteins shared low similarity, implying these proteins may preserve conserved tertiary structure. Null mutants ΔyeeN and ΔyebC both displayed increased swarming and swimming activities, while only ΔyeeN conferred increased tolerance to paraquat. Complementation assays further confirmed functions of these two proteins in motility and paraquat tolerance. Promoter analysis demonstrated that yeeN has its own promoter that can be repressed by oxidative stress. yebC promoter functioned only later growth phase and was not regulated by the test oxidative stress treatments. Protein localization analysis showed that accumulation and stress conditions could lead to polar distribution of YeeN in cytosol, while YebC uniformly distributed in cytosol and became polar distribution in a fragmented manner only at the death growth phase. Furthermore, by investigating the putative regulatory genes that may be involved in yeeN regulation, this study showed that recA could regulate yeeN under oxidative stress treatment. relA could upregulate yeeN expression under normal condition, but may not be involved in yeeN regulation under oxidative stress. On the other hand, hns could downregulate yeeN expression in late growth phase, and may regulate yeeN in flagella biosynthesis. Taken together, these results indicate that YeeN and YebC both function in bacterial motility, while YeeN also plays an important role in oxidative stress responses. This study is the first to uncover common as well as distinct functions of two bacterial DUF28 proteins.