The role of the c-di-GMP specific phosphodiesterase NbdA in NO-induced biofilm dispersal in Pseudomonas aeruginosa

分散是细菌从生物膜过渡到活动生长状态，从而在新的环境中产生新的群落的过程。c-二gmp水平的改变已被证明与许多不同细菌的生物膜分散有关。信号分子一氧化氮（NO）是一种已知的通过刺激c-二gmp降解磷酸二酯酶（PDE）活性诱导生物膜分散的因子。我们最近发现了一种膜锚定蛋白，专门参与机会致病菌铜绿假单胞菌中no诱导的生物膜扩散。no诱导的生物膜分散位点A是由MHYT-GGDEF-EAL融合组成的多结构域蛋白。除了NbdA外，其他几种蛋白质也被证明参与了铜绿假单胞菌生物膜的扩散，包括趋化性传感器BdlA和PDE DipA。为了了解一氧化氮诱导的生物膜分散机制，我们拟利用纯化重组蛋白验证NbdA的感觉功能。基于生物信息学的预测，mhyt结构域被认为是一个通过结合铜离子起作用的气体传感器结构域。紫外-可见和电感耦合等离子体发射光谱结合定点诱变将用于鉴定辅因子和配位氨基酸残基的性质。此外，将进行PDE测定以确定NO是否对PDE活性有刺激作用。由于nbdA敲除突变体在NO处理后无法分散，我们将利用已知的分散位点在生物膜管反应器中进行交叉互补实验，以确定信号转导的顺序。此外，体内膜-链球菌标记蛋白相互作用实验（膜- spine）将用于寻找NbdA的直接相互作用伙伴。先前的数据也表明，在no分散的细胞中，nbdA mRNA水平升高是一种转录调控。我们将利用基于lovv的荧光报告基因融合体研究转录调控，并利用荧光显微镜在生物膜和no分散细胞中监测转录调控。几个已知的转录调控参与no传感将测试其在转录调控中的贡献。在这方面，我们还将测试NO的来源（外源性与内源性）是否对扩散有影响。在这里，我们将利用铜绿假单胞菌突变株，其中包含一个结构完整，但催化活性的亚硝酸盐还原酶。作为SPP群落和研究不依赖于化学、潜在有害信号（如NO）的生物膜扩散的工具，我们将构建基于P. aeruginosa细菌光敏色素的光可控PDE。作为一个长期目标，我们将研究如何感知信号转导成细胞反应，使用荧光报告基因融合和核苷酸捕获化合物的组合来识别信号转导途径中的缺失环节。

Dispersion is a process by which bacteria transit from a biofilm to a motile growth state to spawn novel communities in new locales. Alterations in c-di-GMP levels have been shown to be associated with biofilm dispersal in a number of different bacteria. The signaling molecule nitric oxide (NO) is one factor known to induce biofilm dispersion through stimulation of c-di-GMP degrading phosphodiesterase (PDE) activity. We have recently identified a membrane-anchored protein specifically involved in NO-induced biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa. NO-induced biofilm dispersion locus A is a multidomain protein consisting of a MHYT-GGDEF-EAL fusion. In addition to NbdA, several other proteins were shown to be involved in P. aeruginosa biofilm dispersal, including the chemotaxis transducer BdlA and the PDE DipA. In order to understand the mechanism of NO-induced biofilm dispersal we intend to verify the sensory function of NbdA using purified recombinant protein. Based on bioinformatic predictions the MHYT-domain is proposed to be a gas sensor domain functioning through bound copper ions. UV-Vis and inductively coupled plasma optical emission spectroscopy in combination with site-directed mutagenesis will be used to identify the nature of cofactor and the coordinating amino acid residues. In addition, PDE assays will be performed to establish whether NO has a stimulatory effect on PDE activity. As an nbdA knock-out mutant is unable to disperse upon NO treatment we will perform cross-complementation experiments in biofilm tube reactors using known dispersion loci in order to identify the order of signal transduction. In addition, an in vivo membrane-Strep-tagged protein interaction experiment (membrane-SPINE) will be used to find direct interaction partner(s) to NbdA. Previous data also suggested a transcriptional regulation as nbdA mRNA levels in NO-dispersed cells were elevated. Transcriptional regulation will be investigated employing LOV-based fluorescent reporter fusions and monitored using fluorescence microscopy in biofilms and NO-dispersed cells. Several transcriptional regulators known to be involved in NO-sensing will be tested for their contribution in transcriptional regulation. In this regard we will also test whether the source of NO (exogenous vs. endogenous) has an influence on dispersal. Here we will make use of a P. aeruginosa mutant strain that contains a structurally intact, but catalytically inactive nitrite reductase. As a tool for the SPP community and to study biofilm dispersal independent of chemical, potentially harmful signals (like NO) we will construct a light-controllable PDE based on the bacterial phytochrome of P. aeruginosa. As a long term goal we will investigate how the perceived signal is transduced into a cellular response using a combination of fluorescent reporter gene fusion and nucleotide capture compounds to identify missing links in the signal transduction pathway.