Lu Guang-Tao, Tang Ji-Liang, Wei Guang-Ning, He Yong-Qiang, Chen Bao-Shan
Institute of Molecular genetics, Guangxi University, Nanning 530005, China.
Sheng Wu Gong Cheng Xue Bao. 2004 Jul;20(4):477-83.
Xanthomonas campestris pv. campestris (Xcc), the pathogenic agent of black rot disease in cruciferous plants, produces large amount of extracellular polysaccharide (EPS), which has found wide applications in industry. For the great commercial value of the xanthan gum, many of the genes involved in EPS biosynthesis have been cloned and the mechanism of EPS biosynthesis also has been studied. In order to clone genes involved in EPS biosynthesis, Xcc wild-type strain 8004 was mutagenized with transposon Tn5 gusA5, and a number of EPS-defective mutants were isolated in our previous work. The Tn5 gusA5 inserted sites of these mutants were located by using thermal asymmetric interlaced PCR, and results showed that two EPS-defective mutants were insertion mutants of the gene wxcA which involved in lipopolysaccharide (LPS) biosynthesis. The gene wxcA involved in lipopolysaccharide biosynthesis but dose not extracellular polysaccharide in others' report. wxcA::Tn5 gusA5 mutant 021C12, the polar mutant, was complemented with recombinant plasmid pLATC8570 harboring an intact wxcA gene in this work, but the yield of EPS of the wxcA::Tn5 gusA5 mutant was not restored. In order to identify the function of wxcA gene of Xcc 8004 strain, the gene wxcA was deleted by gene replacement strategy, and the no-polar mutant of wxcA was obtained. DeltawxcA mutant strain, named Xcc 8570, was confirmed by using both PCR and southern analysis. Beside the LPS biosynthesis of deltawxcA mutant was affected, The EPS yield of deltawxcA mutant strain reduced by 50% as compared with the wild-type strain 8004. DeltawxcA mutant could be complemented in trans with the intact wxcA gene, and the EPS yield of the mutant was restored. The combined data showed that wxcA gene not only involved in LPS biosynthesis but also EPS yield in Xcc 8004 strain.
野油菜黄单胞菌野油菜致病变种（Xcc）是十字花科植物黑腐病的病原体，能产生大量胞外多糖（EPS），该多糖在工业上有广泛应用。由于黄原胶具有巨大的商业价值，许多参与EPS生物合成的基因已被克隆，EPS生物合成的机制也得到了研究。为了克隆参与EPS生物合成的基因，用转座子Tn5 gusA5对Xcc野生型菌株8004进行诱变，在我们之前的工作中分离出了许多EPS缺陷型突变体。利用热不对称交错PCR定位了这些突变体中Tn5 gusA5的插入位点，结果表明，两个EPS缺陷型突变体是参与脂多糖（LPS）生物合成的基因wxcA的插入突变体。在其他人的报道中，参与脂多糖生物合成的基因wxcA并不参与胞外多糖的合成。在本研究中，用携带完整wxcA基因的重组质粒pLATC8570对极性突变体wxcA::Tn5 gusA5突变体021C12进行了互补，但wxcA::Tn5 gusA5突变体的EPS产量并未恢复。为了鉴定Xcc 8004菌株wxcA基因的功能，采用基因置换策略缺失了wxcA基因，获得了wxcA的非极性突变体。通过PCR和Southern分析证实了缺失wxcA的突变菌株，命名为Xcc 8570。除了缺失wxcA的突变体的LPS生物合成受到影响外，与野生型菌株8004相比，缺失wxcA的突变菌株的EPS产量降低了50%。缺失wxcA的突变体可以用完整的wxcA基因进行反式互补，突变体的EPS产量得以恢复。综合数据表明，wxcA基因不仅参与Xcc 8004菌株的LPS生物合成，还参与EPS产量的调控。
