Smith T Jarrod, Sondermann Holger, O'Toole George A
Department of Microbiology and Immunology , Geisel School of Medicine at Dartmouth , Room 202 Remsen Building, Hanover , New Hampshire 03755 , United States.
Department of Molecular Medicine, College of Veterinary Medicine , Cornell University , Ithaca , New York 14853 , United States.
ACS Synth Biol. 2018 Nov 16;7(11):2612-2617. doi: 10.1021/acssynbio.8b00278. Epub 2018 Oct 16.
Initial attachment to a surface is a key and highly regulated step in biofilm formation. In this study, we present a platform for reversibly functionalizing bacterial cell surfaces with an emphasis on designing biofilms. We engineered the Lap system of Pseudomonas fluorescens Pf0-1, which is normally used to regulate initial cell surface attachment, to display various protein cargo at the bacterial cell surface and control extracellular release of the cargo in response to changing levels of the second messenger c-di-GMP. To accomplish this goal, we fused the protein cargo between the N-terminal retention module and C-terminal secretion signal of LapA and controlled surface localization of the cargo with natural signals known to stimulate or deplete c-di-GMP levels in P. fluorescens Pf0-1. We show this system can tolerate large cargo in excess of 500 amino acids, direct P. fluorescens Pf0-1 to surfaces it does not typically colonize, and program this microbe to sequester the toxic medal cadmium.
最初附着于表面是生物膜形成过程中的关键且受到高度调控的步骤。在本研究中，我们提出了一个用于对细菌细胞表面进行可逆功能化的平台，重点在于设计生物膜。我们改造了荧光假单胞菌Pf0-1的Lap系统，该系统通常用于调节细胞表面的初始附着，以便在细菌细胞表面展示各种蛋白质负载，并根据第二信使环二鸟苷酸（c-di-GMP）水平的变化来控制负载的细胞外释放。为实现这一目标，我们将蛋白质负载融合在LapA的N端保留模块和C端分泌信号之间，并利用已知可刺激或降低荧光假单胞菌Pf0-1中c-di-GMP水平的天然信号来控制负载的表面定位。我们证明该系统能够容纳超过500个氨基酸的大型负载，引导荧光假单胞菌Pf0-1附着于其通常不会定殖的表面，并使这种微生物能够螯合有毒金属镉。
