National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
Appl Environ Microbiol. 2012 Nov;78(22):8067-74. doi: 10.1128/AEM.01442-12. Epub 2012 Sep 14.
A great deal of research has been done to understand bacterial cell-to-cell signaling systems, but there is still a large gap in our current knowledge because the majority of microorganisms in natural environments do not have cultivated representatives. Metagenomics is one approach to identify novel quorum sensing (QS) systems from uncultured bacteria in environmental samples. In this study, fosmid metagenomic libraries were constructed from a forest soil and an activated sludge from a coke plant, and the target genes were detected using a green fluorescent protein (GFP)-based Escherichia coli biosensor strain whose fluorescence was screened by spectrophotometry. DNA sequence analysis revealed two pairs of new LuxI family N-acyl-L-homoserine lactone (AHL) synthases and LuxR family transcriptional regulators (clones N16 and N52, designated AubI/AubR and AusI/AusR, respectively). AubI and AusI each produced an identical AHL, N-dodecanoyl-L-homoserine lactone (C(12)-HSL), as determined by nuclear magnetic resonance (NMR) and mass spectrometry. Phylogenetic analysis based on amino acid sequences suggested that AusI/AusR was from an uncultured member of the Betaproteobacteria and AubI/AubR was very deeply branched from previously described LuxI/LuxR homologues in isolates of the Proteobacteria. The phylogenetic position of AubI/AubR indicates that they represent a QS system not acquired recently from the Proteobacteria by horizontal gene transfer but share a more ancient ancestry. We demonstrated that metagenomic screening is useful to provide further insight into the phylogenetic diversity of bacterial QS systems by describing two new LuxI/LuxR-type QS systems from uncultured bacteria.
大量的研究已经致力于理解细菌细胞间信号转导系统,但由于大多数自然环境中的微生物没有可培养的代表,我们目前的知识仍然存在很大的差距。宏基因组学是一种从环境样本中未培养的细菌中鉴定新型群体感应(QS)系统的方法。在这项研究中,从森林土壤和焦化厂的活性污泥中构建了 fosmid 宏基因组文库,并使用基于绿色荧光蛋白(GFP)的大肠杆菌生物传感器菌株检测靶基因,其荧光通过分光光度法筛选。DNA 序列分析揭示了两对新的 LuxI 家族 N-酰基-L-高丝氨酸内酯(AHL)合酶和 LuxR 家族转录调节剂(克隆 N16 和 N52,分别命名为 AubI/AubR 和 AusI/AusR)。通过核磁共振(NMR)和质谱分析,确定 AubI 和 AusI 各自产生了相同的 AHL,N-十二烷酰基-L-高丝氨酸内酯(C(12)-HSL)。基于氨基酸序列的系统发育分析表明,AusI/AusR 来自β变形菌的未培养成员,而 AubI/AubR 与 Proteobacteria 中分离株的先前描述的 LuxI/LuxR 同源物非常分支。AubI/AubR 的系统发育位置表明,它们代表一种 QS 系统,不是通过水平基因转移最近从 Proteobacteria 获得的,而是具有更古老的祖先。我们通过描述来自未培养细菌的两种新的 LuxI/LuxR 型 QS 系统,证明宏基因组筛选有助于更深入地了解细菌 QS 系统的系统发育多样性。