Nocker Andreas, Cheung Ching-Ying, Camper Anne K
Center for Biofilm Engineering, Montana State University, 366 EPS Building, P.O. Box 173980, Bozeman, MT 59717-3980, United States.
J Microbiol Methods. 2006 Nov;67(2):310-20. doi: 10.1016/j.mimet.2006.04.015. Epub 2006 Jun 5.
The differentiation between live and dead bacterial cells presents an important challenge in many microbiological applications. Due to the persistence of DNA in the environment after cells have lost viability, DNA-based detection methods cannot differentiate whether positive signals originate from live or dead bacterial targets. We present here a novel chemical, propidium monoazide (PMA), that (like propidium iodide) is highly selective in penetrating only into 'dead' bacterial cells with compromised membrane integrity but not into live cells with intact cell membranes/cell walls. Upon intercalation in the DNA of dead cells, the photo-inducible azide group allows PMA to be covalently cross-linked by exposure to bright light. This process renders the DNA insoluble and results in its loss during subsequent genomic DNA extraction. Subjecting a bacterial population comprised of both live and dead cells to PMA treatment thus results in selective removal of DNA from dead cells. We provide evidence that this chemical can be applied to a wide range of species across the bacterial kingdom presenting a major advantage over ethidium monoazide (EMA). The general application of EMA is hampered by the fact that the chemical can also penetrate live cells of some bacterial species. Transport pumps actively export EMA out of metabolically active cells, but the remaining EMA level can lead to substantial loss of DNA. The higher charge of PMA might be the reason for the higher impermeability through intact cell membranes, thus avoiding DNA loss.
在许多微生物学应用中，区分活细菌细胞和死细菌细胞是一项重大挑战。由于细胞失去活力后DNA仍会在环境中留存，基于DNA的检测方法无法区分阳性信号是来自活细菌靶标还是死细菌靶标。我们在此介绍一种新型化学物质——单叠氮碘化丙锭（PMA），它（与碘化丙锭一样）具有高度选择性，仅能穿透细胞膜完整性受损的“死”细菌细胞，而无法穿透细胞膜/细胞壁完整的活细胞。当嵌入死细胞的DNA中时，光诱导叠氮基团使PMA在强光照射下发生共价交联。这一过程使DNA变得不溶，并导致其在后续基因组DNA提取过程中丢失。因此，对由活细胞和死细胞组成的细菌群体进行PMA处理会导致死细胞的DNA被选择性去除。我们提供的证据表明这种化学物质可应用于细菌界的多种物种，这是相对于单叠氮溴化乙锭（EMA）的一个主要优势。EMA的广泛应用受到限制，因为这种化学物质也能穿透某些细菌物种的活细胞。转运泵会主动将EMA从代谢活跃的细胞中输出，但剩余的EMA水平可能导致大量DNA损失。PMA较高的电荷可能是其更难穿透完整细胞膜的原因，从而避免了DNA损失。
