Study of rapid cell death and pathogenesis of Xanthomonas axonopodis pv. glycines

Abstract

Many eukaryotic organisms practice programmed cell death (PCD) to eliminate damaged or abandoned cells. Recently, some studies suggest that some prokaryotes also possess PCD mechanism. For an example, Xanthomonas axonopodis pv. glycines (Xag) undergoes rapid cell death (RCD) when grown in LB medium for 48 hr but not in starch medium. In order to study the mechanism of RCD, Xag and the RCD-deficient Xanthomonas campestris pv. campestris (Xcc) were compared and no significant differences in growth and survival rates were observed. Using random transposon EZ-Tn5 for mutagenesis, three Xag mutants that initially showed less or no RCD were isolated. However, consistent RCD was no longer observed in further experiments. In LB medium, FeSO4 at low concentration was found to slow down RCD of Xcg, while the cell growth was inhibited at high concentrations. This is in agreement with the result of a previous study showing that FeSO4 can reduce RCD. To further study the Xcg RCD, several mutants were isolated based on the microarray data. Analysis on these mutants revealed that 1) XagcatB, a catalase mutant, has a catalase level which was 50% of that in wild type, but no significant change in RCD was observed, 2) growth of XagiroN, XagfhuA, Xc17iroN, and Xc17fhuA was not affected by iron deficiency in the medium, although it has been shown that FhuA and IroN play some roles in ferric uptake in Xag and Xc, 3) levels of extracellular enzymes were not altered in mutants catB, fhuA, and iroN,and 4) Xag mutants catB, mopB, and fhuA exhibited reduced virulence in causing pustule disease in soybean.

真核生物利用細胞計畫性死亡 (programmed cell death, PCD) 機制控制細胞自殺與消滅受損或不需要的細胞。近年來發現在原核生物中亦有 PCD 機制，例如 Xanthomonas axonopodis pv. glycines (Xag) 的 rapid cell death (RCD) 現象。先前研究發現將 Xag 培養在 LB medium 中48小時後會有 RCD 現象，而在 starch medium 中則否。為探討造成 Xanthomonas RCD 現象之機制，本研究中首先比較 Xag 與 Xanthomonas campestris pv. campestris (Xcc) 在不同培養時期之生長曲線與存活率。Xag 有出現RCD 現象，Xc17則無。然後利用 EZ-Tn5™ <R6Kγori/KAN-2>Tnp Transposome™ kit 隨機突變 Xag基因體，期能找出導致 RCD 現象減弱或消失的相關基因。初步得到3個疑似 RCD 減弱的突變株，但進一步實驗發現其生長與存活情形與野生株相似。前人研究中發現在 LB中添加硫酸亞鐵會影響RCD現象，本研究發現培養基中超過一定濃度的硫酸亞鐵會抑制細菌生長；但低濃度之硫酸亞鐵會使 Xag 生長延遲與減緩RCD。依據 microarray 結果與研究分析，選擇與 catalase 活性及鐵離子吸收相關的 catB、fhuA、iroN 等基因進行突變，並探討其與 RCD 及病原性之相關性。結果顯示，XagcatB 與 XagiroN 之 RCD 現象與野生株相似。Xag 與 Xc17之 iroN 與 fhuA 基因產物可能與鐵離子吸收能力有關；然而在鐵離子缺乏的環境下，XagiroN、XagfhuA、Xc17iroN、Xc17fhuA突變株生長並不會受到影響。XagcatB突變株的 catalase 活性降低，但細菌的 catalase活性高低似乎與 RCD 無相關。測試catB、fhuA、iroN 等突變株的胞外酵素活性後，結果顯示突變株與野生株差異不明顯。進行Xag病原性測試時發現Xag 的 catB、mopB、fhuA突變株的病原性減弱。