Fitness profiling of the Streptococcus mutans genome

描述（由申请人提供）：变形链球菌基因组的适应度分析。变形链球菌形成龋齿需要生物体形成生物膜，成为牙菌斑微生物群落的重要成员，并在牙菌斑内生存（Marsh, 2003）。蛀牙的发生与变形链球菌发酵糖类产生的有机酸对牙釉质的脱矿有关。在酸性适应过程中，变形链球菌通过上调一些蛋白质或脂肪酸在酸性环境中存活下来。来自我们实验室和其他实验室的证据表明，酸适应是一个复杂的多基因过程，目前仅部分了解。随着S.mutans基因组序列的可用性，1963个开放阅读框的注释，以及精确删除单个开放阅读框的方法的发展，分析S.mutans中每个非必需基因成为可能。在这个项目中，我们将研究每个基因对变形链球菌在酸性条件下生长能力的影响。将确定生物体中每个基因对龋齿形成相关事件的贡献。具体目标高通量方法将被定制为可重复地删除整个突变链球菌基因组中的单个基因。每个缺失将被标记为两个独特的DMA序列（18- 20bp）的存在，这样在突变体和野生型群体中每个特定突变株的丰度可以使用这些“条形码”进行评分。具体目标1b。将构建一组标记缺失的基因组，从而形成一个突变链球菌菌株文库，其中包含所有非必需基因的敲除。通过适应度谱分析基因功能的方法将用于确定变形链球菌中每个基因的功能。具体目标2。为了找到可能影响口腔生长的基因，我们将测试突变菌株在体外酸性条件下的生长能力。然后，我们将在确定的条件下使用敲除文库进行适应度分析实验，其中包括：1)在酸性环境中生长的突变菌株文库的共培养，包括浮游和生物膜培养；2)小鼠感染模型，给小鼠喂食高致病性食物。这些实验将作为筛选候选菌株在小鼠龋齿模型中进行单独检查。具体目标3。我们还将研究突变菌株库在其他口腔微生物存在下生长的能力，目的是确定变形链球菌中那些有助于其竞争其他口腔微生物的基因。从这些研究中，我们期望在突变链球菌中鉴定出参与抗酸的基因。与公共卫生相关的是，从这些研究中获得的信息将导致新的见解，并可能为治疗干预提供新的靶点，以了解变形链球菌用于引起人类疾病的特定机制。

DESCRIPTION (provided by applicant): Fitness profiling of the S. mutans genome. Caries formation by S. mutans requires the organism to form a biofilm, become a prominent member of the microbial community of dental plaque and to survive stresses within plaque (Marsh, 2003). The initiation of caries involves demineralization of tooth enamel by organic acids produced by S. mutans fermentation of sugars. S. mutans survives acid environments by the up- regulation of a number of proteins, or, fatty acids in a process referred to as acid-adaptation. Evidence from our laboratory, and others, shows that acid-adaptation is a complex, multi-gene process that is only partially understood at present. With the availability of the S.mutans genome sequence, the annotation of 1,963 open reading frames, and the development of methods precisely to delete individual open reading frames, it is possible to analyze every nonessential gene in S. mutans. In this project, we will examine the effect of each gene on the ability of S. mutans to grow in acidic conditions. The contribution of each gene in the organism to events implicated in caries formation will be determined. Specific Aim 1a. High throughput methods will be tailored to reproducibly delete individual genes in the entire S. mutans genome. Each deletion will be marked by the presence of two unique DMA sequences (18-20 bp) such that abundance of each particular mutant strain in a population of mutants and wild type can be scored using these "bar codes." Specific Aim 1b. A genomic set of marked deletions will be constructed, resulting in a library of S. mutans strains bearing knockouts of all non-essential genes. Methods to analyze gene function by fitness profiling will be used to determine a function for each gene in S. mutans. Specific Aim 2. To find genes that may affect growth in the oral cavity, we will test mutant strains for their ability to grow, in vitro, in acidic conditions. Then, we will use the knockout library in fitness profiling experiments under defined conditions, which will include the following: 1) co-culture of the library of mutant strains growing in acidic environments, including planktonic and biofilm cultures; and, 2) a mouse infection model, in which the mice are fed a highly cariogenic diet. These experiments will be done as screens for candidate strains to be examined individually in the mouse caries model. Specific Aim 3. We will also examine the ability of the library of mutant strains to grow in the presence of additional species of oral microbes, with the aim of identifying those genes in S. mutans that contribute to its ability to out-compete other oral organisms. From these studies, we expect to identify genes in S. mutans that participate in acid-resistance. The relevance to public health is that the information derived from these studies will lead to new insights, and potentially new targets for therapeutic intervention, into specific mechanisms that S. mutans uses to cause disease in humans.