Monteiro P B, Teixeira D C, Palma R R, Garnier M, Bové J M, Renaudin J
Fundo de Defesa da Citricultura (Fundecitrus), 14807-040, VI. Melhado-C.P. 391, Araraquara, São Paulo, Brazil.
Appl Environ Microbiol. 2001 May;67(5):2263-9. doi: 10.1128/AEM.67.5.2263-2269.2001.
Xylella fastidiosa is a gram-negative, xylem-limited bacterium affecting economically important crops (e.g., grapevine, citrus, and coffee). The citrus variegated chlorosis (CVC) strain of X. fastidiosa is the causal agent of this severe disease of citrus in Brazil and represents the first plant-pathogenic bacterium for which the genome sequence was determined. Plasmids for the CVC strain of X. fastidiosa were constructed by combining the chromosomal replication origin (oriC) of X. fastidiosa with a gene which confers resistance to kanamycin (Kan(r)). In plasmid p16KdAori, the oriC fragment comprised the dnaA gene as well as the two flanking intergenic regions, whereas in plasmid p16Kori the oriC fragment was restricted to the dnaA-dnaN intergenic region, which contains dnaA-box like sequences and AT-rich clusters. In plasmid p16K, no oriC sequence was present. In the three constructs, the promoter region of one of the two X. fastidiosa rRNA operons was used to drive the transcription of the Kan(r) gene to optimize the expression of kanamycin resistance in X. fastidiosa. Five CVC X. fastidiosa strains, including strain 9a5c, the genome sequence of which was determined, and two strains isolated from coffee, were electroporated with plasmid p16KdAori or p16Kori. Two CVC isolates, strains J1a12 and B111, yielded kanamycin-resistant transformants when electroporated with plasmid p16KdAori or p16Kori but not when electroporated with p16K. Southern blot analyses of total DNA extracted from the transformants revealed that, in all clones tested, the plasmid had integrated into the host chromosome at the promoter region of the rRNA operon by homologous recombination. To our knowledge, this is the first report of stable transformation in X. fastidiosa. Integration of oriC plasmids into the X. fastidiosa chromosome by homologous recombination holds considerable promise for functional genomics by specific gene inactivation.
桑氏假单胞菌是一种革兰氏阴性、局限于木质部的细菌，可感染具有重要经济价值的作物（如葡萄、柑橘和咖啡）。桑氏假单胞菌的柑橘杂色黄化病（CVC）菌株是巴西这种严重柑橘病害的病原体，也是第一个其基因组序列被测定的植物致病细菌。通过将桑氏假单胞菌的染色体复制起点（oriC）与赋予卡那霉素抗性（Kan(r)）的基因相结合，构建了用于桑氏假单胞菌CVC菌株的质粒。在质粒p16KdAori中，oriC片段包含dnaA基因以及两个侧翼基因间区域，而在质粒p16Kori中，oriC片段局限于dnaA-dnaN基因间区域，该区域含有类似dnaA-box的序列和富含AT的簇。在质粒p16K中，不存在oriC序列。在这三种构建体中，使用了两个桑氏假单胞菌rRNA操纵子之一的启动子区域来驱动Kan(r)基因的转录，以优化桑氏假单胞菌中卡那霉素抗性的表达。用质粒p16KdAori或p16Kori对包括已测定基因组序列的9a5c菌株在内的5个CVC桑氏假单胞菌菌株以及从咖啡中分离的两个菌株进行了电穿孔。当用质粒p16KdAori或p16Kori进行电穿孔时，两个CVC分离株J1a12和B111产生了卡那霉素抗性转化体，但用p16K进行电穿孔时则未产生。对从转化体中提取的总DNA进行的Southern印迹分析表明，在所有测试的克隆中，质粒已通过同源重组整合到rRNA操纵子启动子区域的宿主染色体中。据我们所知，这是桑氏假单胞菌中稳定转化的首次报道。通过同源重组将oriC质粒整合到桑氏假单胞菌染色体中，对于通过特定基因失活进行功能基因组学研究具有很大的前景。
