van Beljouw Sam P B, van der Els Simon, Martens Koen J A, Kleerebezem Michiel, Bron Peter A, Hohlbein Johannes
Laboratory of Biophysics, Wageningen University and Research, Stippeneng 4, 6708 WE Wageningen, The Netherlands. These authors contributed equally.
Phys Biol. 2019 Mar 8;16(3):035001. doi: 10.1088/1478-3975/ab0162.
Lactic acid bacteria (LAB) are frequently used in food fermentation and are invaluable for the taste and nutritional value of the fermentation end-product. To gain a better understanding of underlying biochemical and microbiological mechanisms and cell-to-cell variability in LABs, single-molecule techniques such as single-particle tracking photo-activation localization microscopy (sptPALM) hold great promises but are not yet employed due to the lack of detailed protocols and suitable assays. Here, we qualitatively test various fluorescent proteins including variants that are photoactivatable and therefore suitable for sptPALM measurements in Lactococcus lactis, a key LAB for the dairy industry. In particular, we fused PAmCherry2 to dCas9 allowing the successful tracking of single dCas9 proteins, whilst the dCas9 chimeras bound to specific guide RNAs retained their gene silencing ability in vivo. The diffusional information of the dCas9 without any targets showed different mechanistic states of dCas9: freely diffusing, bound to DNA, or transiently interacting with DNA. The capability of performing sptPALM with dCas9 in L. lactis can lead to a better, general understanding of CRISPR-Cas systems as well as paving the way for CRISPR-Cas based interrogations of cellular functions in LABs.
乳酸菌（LAB）常用于食品发酵，对发酵终产物的风味和营养价值至关重要。为了更好地理解乳酸菌潜在的生化和微生物机制以及细胞间变异性，单分子技术如单粒子追踪光激活定位显微镜（sptPALM）前景广阔，但由于缺乏详细方案和合适的检测方法尚未得到应用。在此，我们对多种荧光蛋白进行了定性测试，包括可光激活的变体，这些变体适用于乳制品行业关键乳酸菌乳酸乳球菌中的sptPALM测量。特别地，我们将PAmCherry2与dCas9融合，成功追踪单个dCas9蛋白，而与特定引导RNA结合的dCas9嵌合体在体内保留了其基因沉默能力。没有任何靶点的dCas9的扩散信息显示出dCas9的不同机制状态：自由扩散、与DNA结合或与DNA短暂相互作用。在乳酸乳球菌中用dCas9进行sptPALM的能力可促进对CRISPR-Cas系统的更好的整体理解，并为基于CRISPR-Cas对乳酸菌细胞功能的研究铺平道路。
