Sivakumar Krishnakumar, Mukherjee Manisha, Cheng Hsin-I, Zhang Yingdan, Ji Lianghui, Cao Bin
Singapore Centre on Environmental Life Science Engineering, Nanyang Technological University, Singapore, 637551, Singapore; Interdisciplinary Graduate School, Nanyang Technological University, Singapore.
Biotechnol Bioeng. 2015 Mar;112(3):512-20. doi: 10.1002/bit.25471. Epub 2014 Oct 21.
Biofilms are the most ubiquitous and resilient form of microbial life on earth. One most important feature of a biofilm is the presence of a self-produced matrix, which creates highly heterogeneous and dynamic microenvironments within biofilms. Redox status in biofilm microenvironments plays a critical role in biofilm development and function. However, there is a lack of non-intrusive tools to quantify extracellular redox status of microenvironments within a biofilm matrix. In this study, using Shewanella oneidensis as a model organism, we demonstrated a novel approach to monitor extracellular redox status in biofilm microenvironments. Specifically, we displayed a redox sensitive fluorescence protein roGFP onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed roGFP as a sensor to quantify the extracellular redox status in the matrix of S. oneidensis biofilms. The fusion of roGFP into BpfA has no negative impacts on cell growth and biofilm formation. Upon exposure to oxidizing agents such as H2 O2 , Ag(+) , and SeO3 (2-) , S. oneidensis BpfA-roGFP cells exhibited a characteristic fluorescence of roGFP. Proteinase treatment assay and super-resolution structured illumination microscopy confirmed the surface localization of BpfA-roGFP. We further used the surface displayed roGFP monitored the extracellular redox status in the matrix at different depths of a biofilm exposed to H2 O2 . This study provides a novel approach to non-invasively monitor extracellular redox status in microenvironments within biofilms, which can be used to understand redox responses of biofilms to environmental perturbations.
生物膜是地球上微生物生命最普遍且具韧性的形式。生物膜的一个最重要特征是存在自我产生的基质，该基质在生物膜内创造出高度异质且动态的微环境。生物膜微环境中的氧化还原状态在生物膜的发育和功能中起关键作用。然而，缺乏非侵入性工具来量化生物膜基质内微环境的细胞外氧化还原状态。在本研究中，我们以嗜铁还原地杆菌作为模式生物，展示了一种监测生物膜微环境中细胞外氧化还原状态的新方法。具体而言，我们通过将氧化还原敏感荧光蛋白roGFP融合到大型表面蛋白BpfA的C末端，将其展示在嗜铁还原地杆菌的细胞表面，并使用表面展示的roGFP作为传感器来量化嗜铁还原地杆菌生物膜基质中的细胞外氧化还原状态。将roGFP融合到BpfA对细胞生长和生物膜形成没有负面影响。暴露于如H2O2、Ag(+)和SeO3(2-)等氧化剂时，嗜铁还原地杆菌BpfA-roGFP细胞表现出roGFP的特征荧光。蛋白酶处理试验和超分辨率结构光照显微镜证实了BpfA-roGFP的表面定位。我们进一步使用表面展示的roGFP监测了暴露于H2O2的生物膜不同深度基质中的细胞外氧化还原状态。本研究提供了一种非侵入性监测生物膜内微环境中细胞外氧化还原状态的新方法，可用于了解生物膜对环境扰动的氧化还原反应。
