Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida/IFAS, 700 Experiment Station Road, Lake Alfred, FL 33850, USA.
Appl Environ Microbiol. 2010 Apr;76(7):2234-42. doi: 10.1128/AEM.02897-09. Epub 2010 Jan 29.
Xanthomonas citri subsp. citri is the causal agent of citrus canker, which has a significant impact on citrus production. In this study, we characterized the galU gene of X. citri subsp. citri. Two galU mutants (F6 and D12) were identified in an X. citri subsp. citri EZ-Tn5 <R6Kgammaori/KAN-2> Tnp transposon library. Rescue cloning, sequence analysis, and Southern blot analysis indicated that both of these mutants had a single copy of the EZ-Tn5 transposon inserted in galU in the chromosome. Further study showed that galU was required for biosynthesis of extracellular polysaccharides (EPS; xanthan gum) and capsular polysaccharide (CPS) and biofilm formation. Mutation of galU resulted in a loss of pathogenicity for grapefruit. The loss of pathogenicity of a galU mutant resulted from its inability to grow in planta rather than from the effect on virulence genes. Quantitative reverse transcription-PCR assays indicated that mutation of galU did not impair the expression of key virulence genes, such as pthA of X. citri subsp. citri. Although D12 had a growth rate similar to that of the wild-type strain in nutrient broth, no D12 population became established in the intercellular spaces of citrus leaves. Coinoculation of a galU mutant with the wild-type strain did not promote growth of the galU mutant in planta. Defects in EPS and CPS production, pathogenicity, and growth in planta of the galU mutant were complemented to the wild-type level using plasmid pCGU2.1 containing an intact galU gene. These data indicate that the galU gene contributes to X. citri subsp. citri growth in intercellular spaces and is involved in EPS and CPS synthesis and biofilm formation.
桔亚属柠檬欧文氏菌的 galU 基因的特征
桔亚属柠檬欧文氏菌是柑桔溃疡病的致病因子，对柑桔生产有重大影响。在本研究中，我们对桔亚属柠檬欧文氏菌的 galU 基因进行了特征描述。在桔亚属柠檬欧文氏菌 EZ-Tn5 <R6Kgammaori/KAN-2> Tnp 转座子文库中鉴定出两个 galU 突变体（F6 和 D12）。拯救克隆、序列分析和 Southern blot 分析表明，这两个突变体在染色体上 galU 基因中都只有一个 EZ-Tn5 转座子的单拷贝插入。进一步的研究表明，galU 基因对于胞外多糖（黄原胶）和荚膜多糖（CPS）的生物合成和生物膜形成是必需的。galU 突变导致葡萄柚的致病性丧失。galU 突变体的致病性丧失是由于其在植物体内无法生长，而不是对毒力基因的影响。定量逆转录-PCR 分析表明，galU 突变不会损害关键毒力基因（如桔亚属柠檬欧文氏菌的 pthA）的表达。尽管 D12 在营养肉汤中的生长速率与野生型菌株相似，但在柑桔叶片的细胞间隙中没有建立 D12 种群。galU 突变体与野生型菌株共同接种不会促进 galU 突变体在植物体内的生长。用含有完整 galU 基因的质粒 pCGU2.1 可将 EPS 和 CPS 产生、致病性和植物体内生长的 galU 突变体缺陷部分互补至野生型水平。这些数据表明，galU 基因有助于桔亚属柠檬欧文氏菌在细胞间隙中的生长，并且参与 EPS 和 CPS 的合成和生物膜的形成。
