Characterizing regulatory RNAs in oral polymicrobial community

性状（由申请方提供）：伴放线菌聚集杆菌（Aa）是一种革兰氏阴性菌，存在于人类龈沟中，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会对来自其邻居的线索做出反应，例如戈登链球菌（Sg），改变其饮食[3]并避免宿主免疫防御[4]。虽然这些相互作用被认为是重要的Aa在口腔中的生存，介导这些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，包括小的非编码调节RNA（sRNA）和代谢物传感核糖开关，来改变基因表达，但没有一个在Aa中被表征。这导致了Aa表达调节RNA以改变基因表达以响应环境变化和来自邻近细菌的线索的假设。最初，使用北方印迹分析来检测23种预测sRNA的表达（http：//www.oralgen.lanl.gov/）。共检测到12个差异表达的sRNA。我的特点是第一个赖氨酸核糖开关和新的赖氨酸转运蛋白，LysT，在Aa。由于核糖开关有一个长的半衰期和高水平的表达，我假设赖氨酸核糖开关在细胞中有另一个作用，作为一个反式作用的sRNA。在第一个目标，我提出的实验，以确定反式调节目标的赖氨酸核糖开关。我还将鉴定基因表达中的赖氨酸依赖性变化。该提议的第二个目的建立在第一个目的的基础上，但采用全局方法来鉴定具有Aa和Sg的多微生物生物膜群落中的调节RNA。这种基于技术和训练的方法将使用下一代深度测序来映射转录起始位点和来自Aa和Sg的转录物，以及鉴定由两种细菌表达的差异调节的转录物和候选调节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的转录调节组;确定两种细菌在多微生物群落中表达的调节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Aa和Sg的遗传调控研究。在整个拟议的工作中，技术将在生物信息学，生物化学，遗传学和分子生物学学习，并将为进一步的研究工作提供一个很好的基础。 公共卫生相关性：多种细菌之间的相互作用对于口腔组织的定殖和细菌对宿主免疫的防御是重要的。该提案的目标是表征口腔中介导细菌多物种相互作用的调控机制，并开发新技术，为研究界提供强大的工具。这些研究将帮助我们更好地了解口腔细菌如何在导致人类疾病的多种微生物群落中茁壮成长。

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. PUBLIC HEALTH RELEVANCE: Interactions between multiple species of bacteria are important for colonization of oral tissues and bacterial defenses to host immunity. The goal of this proposal is to characterize the regulatory mechanisms mediating bacterial multispecies interactions in the mouth, and develop new technology that will provide powerful tools for the research community. These studies will help us better understand how oral bacteria thrive in polymicrobial communities that cause human disease.