Targeted gene disruption by homologous recombination, has been widely used in mycobacterium species to understand the genetic basis of virulence and persistence in the host and to develop efficacious potential live vaccines. However, in slow growing pathogenic mycobacteria as Mycobacterium avium subsp paratuberculosis (MAP), these methods have been inefficient, in part due to the low frequency of legitimate homologous recombination. Another feature of mycobacteria is the low efficiency of transformation; therefore, some years ago, a phage-mediated transduction process was developed to introduce DNA into mycobacteria. This strategy is very efficient, due to the high rate of infection of the phage. This report describes a genetic method for the generation of targeted deletion mutations in MAP by allelic exchange using in vitro-generated specialized transducing mycobacteriophages, which does not require the critical packaging step and that could also be applied to other mycobacteria. We provide a detailed gene deletion methodology and demonstrate the use of this genetic system by deleting the mce4 operon of MAP. Finally, our results showed that the deletion of mce4 in MAP induces triacylglycerol accumulation; alter morphology and aggregation in liquid culture.

通过同源重组的靶向基因破坏已广泛用于分枝杆菌属物种中，以了解宿主中毒力和持久性的遗传基础，并开发出有效的潜在活疫苗。然而，在缓慢增长的致病性分枝杆菌中，如鸟分枝杆菌副结核亚种（MAP），这些方法效率低下，部分原因是合法同源重组的频率较低。分枝杆菌的另一个特征是转化效率低。因此，几年前，开发了一种噬菌体介导的转导过程以将DNA引入分枝杆菌中。由于噬菌体的高感染率，该策略非常有效。该报告描述了一种遗传方法，该方法通过使用体外产生的专门的转导分枝杆菌噬菌体，通过等位基因交换，在MAP中产生靶向缺失突变，该方法不需要关键的包装步骤，也可以应用于其他分枝杆菌。我们提供了详细的基因删除方法，并通过删除mce展示了该遗传系统的使用MAP的4个操纵子。最后，我们的结果表明，MAP中mce 4的缺失诱导了三酰基甘油的积累；改变液体培养物中的形态和聚集。