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

扩散是细菌从生物膜过渡到可移动的生长状态，以在新的地点产生新的群落的过程。C-di-GMP水平的变化已被证明与生物膜在许多不同细菌中的扩散有关。信号分子一氧化氮(NO)是一种已知的通过刺激c-di-GMP降解磷酸二酯酶(PDE)活性来诱导生物膜分散的因素。我们最近在条件致病菌铜绿假单胞菌中发现了一种膜锚定蛋白，它与NO诱导的生物膜扩散有关。NO诱导的生物膜分散位点A是由MHYT-GGDEF-EAL融合而成的多域蛋白。除了NBDA，其他几种蛋白也参与了铜绿假单胞菌生物膜的扩散，包括趋化转导蛋白BdlA和PDE DipA。为了了解NO诱导生物膜分散的机制，我们打算用纯化的重组蛋白来验证NBDA的感觉功能。基于生物信息学预测，MHYT结构域被认为是一个通过结合铜离子发挥作用的气敏结构域。UV-Vis和电感耦合等离子体光发射光谱结合定点突变将被用来鉴定辅因子的性质和配位氨基酸残基。此外，还将进行PDE分析，以确定NO是否对PDE活性有刺激作用。由于NBDA基因敲除突变体在没有处理的情况下不能分散，我们将使用已知的分散位置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，还将通过活体膜-链标记蛋白相互作用实验(膜-脊柱)寻找与NBDA直接相互作用的伙伴(S)。先前的数据也表明，当非分散细胞中NBDA mRNA水平升高时，转录调控。将使用基于LOV的荧光报告融合来研究转录调控，并使用荧光显微镜在生物膜和非分散细胞中进行监测。几个已知参与NO感应的转录调控因子将被测试它们在转录调控中的作用。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，我们将利用一株铜绿假单胞菌突变株，该突变株含有结构完整但催化活性不强的亚硝酸还原酶。作为SPP群落的一个工具，并研究不依赖于化学、潜在有害信号(如NO)的生物膜扩散，我们将基于铜绿假单胞菌的细菌光敏色素构建一个光可控的PDE。作为一个长期目标，我们将使用荧光报告基因融合和核苷酸捕获化合物的组合来研究感知信号如何转化为细胞反应，以识别信号转导途径中缺失的环节。

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.