Study of rapid cell death in Xanthomonas campestrispv. glycines

Abstract

Xanthomonas campestris pv. glycines (Xcg) exhibits post-exponential rapid cell death (RCD) in LB medium. To investigate the mechanism of Xcg RCD, growth and survival rate of Xcg and X. campestris pv. campestris (Xcc) was first compared. The results indicated that RCD occurs in Xcg but not in Xcc. Random transposon mutagenesis using EZ-Tn5 was employed to isolate 4000 mutants; however, growth of all the insertional mutant strains in LB medium was found to be the same as that of the wild type strain. Based on previous findings and my results of two-dimensional gel electrophoresis (2D-GE) of the total cellular proteins and microarray analysis of the transcriptomes, several genes were suspected to be involved in RCD, which included pda, clp, glyAB, mopB, recA, genes encoding unknown protein 1318 and unknown protein 0249. A mutant of each of these genes was created separately by insertional inactivation. Growth curve in LB medium and XOLN medium, survival, and extracellular enzyme activity were assayed for these mutants. Results showed that i) glyAB, recA, unknown protein 1318, and unknown protein 0249 mutants exhibited the same growth rates similar to that of the wild-type Xcg, nor the RCD was affected, ii) enhanced RCD was observed in mopB mutant strain, and iii) secretion of extracellular enzymes was significantly affected in clp and mopB mutants but not in pda, glyAB, recA, unprotein 1318 and unprotein 0249 mutant strains. To understand the effect of salts on Xcg growth, different concentrations of NaCl, FeSO4, MgSO4, NiSO4and FeCl3 were added to the medium. Results showed that the addition of NaCl, MgSO4 and FeCl3 did not affect the growth and survival of Xcg, while addition of different concentrations of NiSO4 and FeSO4 slowed down Xcg RCD, indicating that Ni+2 and Fe+2 ions play an important role in RCD. Furthermore, glutathione (GSH) and glucose were found to suppress Xcg RCD, but the RCD was not affected by addition of nalidixic acid and dimethylsulfoxide (DMSO).

將Xanthomonascampestris pv. glycines (Xcg) 培養在 LB medium 中，生長進入靜止期後，細胞會有快速死亡 (rapid cell death, RCD) 的現象。為探討造成Xcg RCD現象之機制，本研究中首先觀察比較 Xcg與X. campestris pv. campestris (Xcc)之生長，發現 RCD 是 Xcg 特有現象。利用 EZ-Tn5™ <R6Kγori/KAN-2> Tnp Transposome™ Kit 進行基因體之任意突變，尚未能找出與 RCD 現象相關的基因。依據two-dimensional gelelectrophoresis (2D) 與 microarray 實驗方法，分析不同時期之蛋白質表現與 RNA 表現量差異之基因，以及過去研究文獻之報導，推測可能參與 RCD 現象的基因為 pda、clp、glyAB、mopB、recA、unknown protein 1318、unknown protein 0249。針對 Xcg pda、clp、glyAB、mopB、recA、unknown protein 1318、unknown protein 0249 等基因進行突變，構築獲得7個基因突變株。測試各突變株在 LB 與 XOLN medium中之生長曲線、存活菌數與胞外酵素活性之結果顯示，pda、clp、glyAB、recA、unprotein 1318與unprotein 0249 基因突變後並不會影響 Xcg 的 RCD 現象。但 mopB 之突變會提早 RCD 現象產生。進一步研究發現，pda、glyAB、recA、unprotein 1318與unprotein 0249之突變不影響 Xcg 胞外酵素的分泌。而 clp 與 mopB 之突變會明顯影響胞外酵素活性。為了解離子化合物對於 Xcg 之生長效應，在 LB 培養液中分別加入不同濃度之 NaCl、FeSO4、MgSO4、NiSO4以及 FeCl3。結果顯示，在 LB 培養基液中加入不同濃度之 NaCl、MgSO4以及 FeCl3，對於 Xcg生長與存活菌數沒有明顯影響。然而，加入不同濃度 NiSO4與 FeSO4後， Xcg 的 RCD 現象明顯趨緩，顯示 Ni＋2 與 Fe＋2 離子在 Xcg 的 RCD 機制中扮演重要的角色。此外，glutathione (GSH) 與葡萄糖也有抑制 Xcg RCD 的效應。但nalidixic acid與dimethylsulfoxide (DMSO) 則未有抑制 RCD 的功能。