Characterizing regulatory RNAs in oral polymicrobial community

描述(由申请人提供)：伴生放线杆菌(AA)是一种革兰氏阴性共生菌，是一种牙周条件致病菌。牙龈缝隙是一个微生物丰富的环境，AA会对邻居的提示做出反应，如戈登链球菌(Sg)，改变它的饮食[3]，避免宿主免疫防御[4]。虽然这些相互作用被认为对口腔中AA的生存很重要，但调节这些物种间相互作用的遗传调控机制在很大程度上是未知的。几乎所有的细菌都利用非编码RNA，包括小的非编码调控RNA(SRNAs)和代谢物感应核糖开关，来改变基因表达，但还没有一个在AA中被表征。这导致了一种假设，即AA表达调控RNA，以改变基因表达，以响应环境的变化和来自邻近细菌的线索。首先，使用Northern印迹分析来检测23个预测的sRNA(http://www.oralgen.lanl.gov/).)的表达共检测到12个差异表达的sRNA。我已经鉴定了AA中的第一个赖氨酸核糖开关和新的赖氨酸转运蛋白Lyst。由于核糖开关有很长的半衰期，并且在高水平表达，我推测赖氨酸核糖开关在细胞中作为反式作用的sRNA具有另一个作用。在第一个目标中，我提议进行实验，以确定赖氨酸核糖开关的反式调控靶标。我还将确定赖氨酸依赖的基因表达变化。该提案的第二个目标建立在第一个目标的基础上，但采取了一种全球方法来识别具有AA和Sg的多微生物生物膜群落中的调控RNA。这一基于技术和培训的方法将使用下一代深度测序来绘制Aa和Sg的转录起始点和转录本，以及识别这两种细菌表达的差异调控转录本和候选调控RNA，包括sRNA、核糖开关和反义RNA。这项研究将提供第一个多物种转录调节组；鉴定多微生物群落中两个细菌表达的调控RNA，并将为口腔微生物学研究界提供重要信息，这将导致未来对AA和Sg基因调控的研究。在整个拟议的工作中，将学习生物信息学、生物化学、遗传学和分子生物学的技术，并将为进一步的研究努力提供良好的基础。

DESCRIPTION (provided by applicant): Aggregatibacter actinomycetemcomitans (Aa) is a Gram-negative commensal of the human gingival crevice and is an opportunistic periodontal pathogen. The gingival crevice is a microbe-rich environment, and Aa responds to cues from its neighbors, such as Streptococcus gordonii (Sg), altering its diet [3] and avoiding host immune defenses [4]. While these interactions are thought to be important for the survival of Aa in the oral cavity, the genetic regulatory mechanisms mediating these interspecies interactions are largely unknown. Nearly all bacteria utilize non-coding RNAs, including small non-coding regulatory RNAs (sRNAs) and metabolite sensing riboswitches, to alter gene expression but none have been characterized in Aa. This led to the hypothesis that Aa expresses regulatory RNAs to alter gene expression in response to changes in the environment and cues from neighboring bacteria. Initially, Northern blot analysis was used to detect the expression of 23 predicted sRNAs (http://www.oralgen.lanl.gov/). 12 differentially expressed putative sRNAs were detected. I have characterized the first lysine riboswitch and the novel lysine transporter, LysT, in Aa. Since the riboswitch has a long half-life and is expressed at high levels, I hypothesize that the lysine riboswitch has another role in the cell as a trans-acting sRNA. In the first aim I propose experiments to determine trans regulatory targets for the lysine riboswitch. I will also identify lysine-dependent changes in gene expression. The second aim of this proposal builds off of the first aim, but takes a global approach to identify regulatory RNAs in a polymicrobial biofilm community with Aa and Sg. This technology and training based approach will use next generation deep sequencing to map transcriptional start sites and transcripts from Aa and Sg, as well as identify differentially regulated transcripts and candidate regulatory RNAs expressed by both bacteria, including sRNAs, riboswitches, and antisense RNAs. This study will provide the first multispecies transcriptional regulome; identifying regulatory RNAs expressed by two bacteria in a polymicrobial community, and will provide important information for the oral microbiology research community that will lead to future studies of genetic regulation in both Aa and Sg. Throughout the proposed work, techniques will be learned in bioinformatics, biochemistry, genetics, and molecular biology, and will provide an excellent foundation for further research endeavors.