Citrus Research and Education Center, Department of Microbiology and Cell Science, University of Florida, IFAS, Lake Alfred, 33850, USA.
BMC Microbiol. 2012 Mar 9;12:31. doi: 10.1186/1471-2180-12-31.
The Gram-negative bacterium Xanthomonas citri subsp. citri (Xac) causes citrus canker, one of the most destructive diseases of citrus worldwide. In our previous work, a transposon mutant of Xac strain 306 with an insertion in the XAC3110 locus was isolated in a screening that aimed at identifying genes related to biofilm formation. The XAC3110 locus was named as bdp24 for biofilm-defective phenotype and the mutant was observed to be affected in extracellular polysaccharide (EPS) and lipopolysaccharide (LPS) biosynthesis and cell motility. In this study, we further characterized the bdp24 (XAC3110) gene (designated as gpsX) using genetic complementation assays and expanded the knowledge about the function of the gpsX gene in Xac pathogenesis by investigating the roles of gpsX in EPS and LPS production, cell motility, biofilm formation on host leaves, stress tolerance, growth in planta, and host virulence of the citrus canker bacterium.
The gpsX gene encodes a putative glycosyltransferase, which is highly conserved in the sequenced strains of Xanthomonas. Mutation of gpsX resulted in a significant reduction of the amount of EPS and loss of two LPS bands visualized on sodium dodecylsulphate- polyacrylamide gels. Biofilm assays revealed that the gpsX mutation affected biofilm formation by Xac on abiotic and biotic surfaces. The gpsX mutant showed delayed bacterial growth and caused reduced development of disease symptoms in susceptible citrus leaves. The gpsX mutant was more sensitive than the wild-type strain to various stresses, including the H(2)O(2) oxidative stress. The mutant also showed attenuated ability in cell motility but not in flagellar formation. Quantitative reverse transcription-PCR assays indicated that mutation of gpsX did not affect the expression of virulence genes such as pthA in Xac strain 306. The affected phenotypes of the gpsX mutant could be complemented to wild-type levels by the intact gpsX gene.
Taken together, our data confirm that the gpsX gene is involved in EPS and LPS synthesis and biofilm formation in Xac and suggest that the gpsX gene contributes to the adaptation of Xac to the host microenvironments at early stage of infection and thus is required for full virulence on host plants.
革兰氏阴性细菌柑橘溃疡病菌（Xanthomonas citri subsp. citri，Xac）引起的柑橘溃疡病是世界范围内对柑橘危害最严重的疾病之一。在我们之前的工作中，通过筛选与生物膜形成相关的基因，分离到了一个在 Xac 菌株 306 的 XAC3110 基因座插入转座子突变体。XAC3110 基因座被命名为 bdp24，代表生物膜缺陷表型，突变体被观察到在外生多糖（EPS）和脂多糖（LPS）生物合成和细胞运动方面受到影响。在这项研究中，我们使用遗传互补实验进一步表征了 bdp24（XAC3110）基因（命名为 gpsX），并通过研究 gpsX 在 EPS 和 LPS 产生、细胞运动、宿主叶片生物膜形成、应激耐受、植物生长和柑橘溃疡病菌宿主毒力方面的作用，扩展了 gpsX 基因在 Xac 发病机制中的功能知识。
gpsX 基因编码一个假定的糖基转移酶，在已测序的黄单胞菌菌株中高度保守。gpsX 突变导致 EPS 量显著减少，并且在十二烷基硫酸钠-聚丙烯酰胺凝胶上可见两个 LPS 带缺失。生物膜实验表明，gpsX 突变影响了 Xac 在非生物和生物表面的生物膜形成。gpsX 突变体在敏感柑橘叶片中的细菌生长延迟，并导致疾病症状的发展减少。与野生型菌株相比，gpsX 突变体对各种应激，包括 H（2）O（2）氧化应激更敏感。该突变体在细胞运动方面也表现出减弱的能力，但在鞭毛形成方面没有。定量逆转录-PCR 分析表明，gpsX 突变不影响 Xac 菌株 306 中 pthA 等毒力基因的表达。完整的 gpsX 基因可以将 gpsX 突变体的表型恢复到野生型水平。
综上所述，我们的数据证实 gpsX 基因参与了 Xac 的 EPS 和 LPS 合成以及生物膜形成，并表明 gpsX 基因有助于 Xac 在感染早期适应宿主微环境，因此是在宿主植物上完全毒力所必需的。
