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首页> 外文期刊>Applied Microbiology >Development of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag Proteins
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Development of Strong Anaerobic Fluorescent Reporters for Clostridium acetobutylicum and Clostridium ljungdahlii Using HaloTag and SNAP-tag Proteins

机译：用卤橡胶和捕获标签蛋白的梭菌乙酰丁基糖尿布和梭菌菌株的强厌氧荧光报道器的开发

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One of the biggest limitations in the study and engineering of anaerobic Clostridium organisms is the lack of strong fluorescent reporters capable of strong and real-time fluorescence. Recently, we developed a strong fluorescent reporter system for Clostridium organisms based on the FAST protein. Here, we report the development of two new strong fluorescent reporter systems for Clostridium organisms based on the HaloTag and SNAP-tag proteins, which produce strong fluorescent signals when covalently bound to fluorogenic ligands. These new fluorescent reporters are orthogonal to the FAST ligands and to each other, allowing for simultaneous labeling and visualization. We used HaloTag and SNAP-tag to label the strictly anaerobic organisms Clostridium acetobutylicum and Clostridium ljungdahlii . We have also identified a new strong promoter for protein expression in C. acetobutylicum , based on the phosphotransacetylase gene ( pta ) from C. ljungdahlii . Furthermore, the HaloTag and the SNAP-tag, in combination with the previously described FAST system, were successfully used to measure cell populations in bacterial mixed cultures and showed the simultaneous orthogonal labeling of HaloTag and SNAP-tag together with the FAST protein reporter. Finally, we show the expression of recombinant fusion protein of FAST and the ZapA division protein (from C. acetobutylicum ) in C. ljungdahlii. The availability of multiple strong fluorescent reporters is a major addition to the genetic toolkit of Clostridium and other anaerobes that will lead to better understanding of these unique organisms.IMPORTANCE Up to this point, assays and methods involving fluorescent reporter proteins were unavailable or limited in Clostridium organisms and other strict anaerobes. Green fluorescent protein (GFP), mCherry, and flavin-binding proteins (and their derivatives) have been used only in a few clostridia with limited success and yielded low fluorescence compared to aerobic microbial systems. Recently, we reported a new strong fluorescent reporter system based on the FAST protein as a first step in expanding the fluorescence-based reporters for Clostridium and other anaerobic microbial platforms. Additional strong orthogonal fluorescent proteins, with distinct emission spectra are needed to allow for (i) multispecies tracking within the growing field of microbial cocultures and microbiomes, (ii) protein localization and tracking in anaerobes, and (iii) identification and development of natural and synthetic promoters, ribosome-binding sites (RBS), and terminators for optimal protein expression in anaerobes. Here, we present two new strong fluorescent reporter systems based on the HaloTag and SNAP-tag proteins.
机译：厌氧梭菌生物的研究和工程中最大的局限性之一是缺乏能够强大荧光的强大荧光记者。最近，我们对基于快速蛋白质的梭菌生物体开发了强大的荧光报道系统。在这里，我们报告了基于卤素和捕获标签蛋白的梭菌生物体的两个新的强荧光报道系统的开发，其在共价结合到荧光配体时产生强荧光信号。这些新的荧光报道器与快速配体和彼此正交，允许同时标记和可视化。我们使用了哈拉焦和捕捉标签来标记严格的厌氧生物梭菌乙酸纤维素和梭菌菌株Ljungdahlii。我们还确定了一种新的强大的促进剂，用于基于来自C.Ljungdahlii的磷酸氯酰基酶基因（PTA）的C.乙酰丁酯中的蛋白质表达。此外，Halotag和卡扣标签与先前描述的快速系统组合用于测量细菌混合培养物中的细胞群，并与快速蛋白质报告者一起显示Halotag和Snap-Tag的同时正交标记。最后，我们展示了C.Ljungdahlii中的快速和Zapa分裂蛋白（来自C.乙酰丁基）的重组融合蛋白的表达。多种强荧光报告者的可用性是梭菌和其他厌氧人的遗传工具的重大增长，这将导致更好地了解这些独特的生物体。分析到这一点，涉及荧光报道蛋白的测定和方法在梭菌中不可用或有限生物和其他严格的厌氧人。绿色荧光蛋白（GFP），MCHERRY和黄蛋白结合蛋白（及其衍生物）仅在几个蛋白质中仅使用，其成功有限，与有氧微生物系统相比产生低荧光。最近，我们报告了一种基于快速蛋白质的新型强荧光报道系统，作为扩展梭菌和其他厌氧微生物平台的荧光的报告的第一步。需要具有明显发射光谱的额外强正交荧光蛋白，以允许（i）在微生物培养和微生物体和微生物体的越来越多的领域内进行多数跟踪，（ii）蛋白质定位和跟踪，（iii）自然的识别和发展合成促进剂，核糖体结合位点（RBS）和终止剂，用于在厌氧中的最佳蛋白表达。在这里，我们介绍了基于Halotag和Snap-Tag蛋白的两种新的强荧光报道系统。

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