Xanthomonas campestrispv. glycines 細胞快速死亡現象之探討

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).