Fitness profiling of the Streptococcus mutans genome

描述（由申请人提供）：S.变异体基因组龋病形成由S.变形杆菌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，并在牙菌斑内的压力下存活（Marsh，2003）。龋病的发生涉及由S.糖的变形菌发酵。S.变形菌通过在称为酸适应的过程中上调许多蛋白质或脂肪酸而在酸性环境中存活。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。随着变形链球菌基因组序列的获得、1,963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，分析变形链球菌中的每个非必需基因成为可能。变异人在本项目中，我们将研究每个基因对S。变形菌在酸性条件下生长。将确定生物体中每个基因对涉及龋齿形成的事件的贡献。具体目标1a。高通量方法将被定制为可重复地删除整个S中的单个基因。变异体基因组每个缺失将通过两个独特的DMA序列（18-20 bp）的存在来标记，使得可以使用这些“条形码”对突变体和野生型群体中每个特定突变体菌株的丰度进行评分。“具体目标1b。将构建一组标记缺失的基因组，产生S.所有非必需基因被敲除的变异株。通过适合度分析基因功能的方法将用于确定S.变异人具体目标2。为了找到可能影响口腔生长的基因，我们将测试突变株在体外酸性条件下生长的能力。然后，我们将在限定条件下在适合性分析实验中使用敲除文库，其将包括以下：1）在酸性环境中生长的突变株文库的共培养，包括嗜酸性和生物膜培养物;和2）小鼠感染模型，其中小鼠被喂食高度致龋饮食。这些实验将作为候选菌株的筛选来进行，以在小鼠龋齿模型中单独检查。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的生长能力，目的是鉴定S。这些变异体有助于其竞争其他口腔生物的能力。通过这些研究，我们希望能够鉴定出S.参与抗酸性的变异体。与公共卫生的相关性在于，从这些研究中获得的信息将导致对S.变异体用来引起人类疾病

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.