An <Emphasis Type="Italic">ipdC</Emphasis> gene knock-out of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> strain SM and its implications on indole-3-acetic acid biosynthesis and plant growth promotion

The indole-3-pyruvate decarboxylase gene (ipdC), coding for a key enzyme of the indole-3-pyruvic acid pathway of IAA biosynthesis in Azospirillum brasilense SM was functionally disrupted in a site-specific manner. This disruption was brought about by group II intron-based Targetron gene knock-out system as other conventional methods were unsuccessful in generating an IAA-attenuated mutant. Intron insertion was targeted to position 568 on the sense strand of ipdC, resulting in the knock-out strain, SMIT568s10 which showed a significant (∼50%) decrease in the levels of indole-3-acetic acid, indole-3-acetaldehyde and tryptophol compared to the wild type strain SM. In addition, a significant decrease in indole-3-pyruvate decarboxylase enzyme activity by ∼50% was identified confirming a functional knock-out. Consequently, a reduction in the plant growth promoting response of strain SMIT568s10 was observed in terms of root length and lateral root proliferation as well as the total dry weight of the treated plants. Residual indole-3-pyruvate decarboxylase enzyme activity, and indole-3-acetic acid, tryptophol and indole-3-acetaldehyde formed along with the plant growth promoting response by strain SMIT568s10 in comparison with an untreated set suggest the presence of more than one copy of ipdC in the A. brasilense SM genome.     

Targeted gene disruption in Francisella tularensis by group II introns

Francisella tularensis is a highly infectious Gram-negative bacterium that is the causative agent of tularemia. Very little is known about the molecular mechanisms responsible for F. tularensis virulence, in part due to the paucity of genetic tools available for the study of F. tularensis. We have developed a gene knockout system for F. tularensis that utilizes retargeted mobile group II introns, or “targetrons”. These targetrons disrupt both single and duplicated target genes at high efficiency in three different F. tularensis subspecies. Here we describe in detail the targetron-based method for insertional mutagenesis of F. tularensis genes, which should facilitate a better understanding of F. tularensis pathogenesis. Group II introns can be adapted to inactivate genes in bacteria for which few genetic tools exist, thus providing a powerful tool to study the genetic basis of bacterial pathogenesis.     

L1.LtrB内含子编码蛋白反转录结构域关键催化位点分析及功能验证

【目的】筛选影响Ll.LtrB内含子编码蛋白(Intron encoded protein,IEP)反转录功能的关键催化位点,并获得无反转录活性的IEP突变体。【方法】首先,利用NCBI数据库,通过序列比对及同源建模方法筛选影响IEP反转录功能的关键氨基酸催化位点;然后,对筛选获得的关键催化位点进行定点突变,同时以Targetron载体为模板,构建无反转录功能的突变型Targetron打靶系统;最后,以大肠杆菌lacZ基因为例,体内验证IEP突变体的功能及其对Ⅱ型内含子"归巢"效率的影响。【结果】筛选到C164和G214两个位点是影响内含子编码蛋白反转录功能的关键氨基酸残基,并获得C164K和G214W两个突变体。体内功能分析表明,此两个位点突变完全失活了Ⅱ型内含子的"归巢"功能。【结论】筛选并获得了失活反转录功能的Ll.LtrB内含子编码蛋白突变体,为深入研究Ⅱ型内含子的结构和"归巢"机理奠定了基础。