Systematic Phenotypic Analysis of the Gram-positive Envelope

描述（由申请人提供）：细菌细胞由一个与环境直接和持续接触的信封包裹。因此，包膜是细菌首先感知环境波动（如渗透压变化）的部位。此外，包膜的成分介导其他细菌之间的交流以及细菌与人类宿主之间的共生或致病相互作用。这些相互作用在人类肠道微生物群等复杂环境中发挥着关键作用。最后，许多广泛使用的最成功的抗生素靶向包膜（例如，青霉素和万古霉素），并且具有细胞质靶点的抗生素的疗效经常因包膜通透性问题而降低。由于这些原因，信封是一个激烈的研究重点领域。研究单个基因产物或途径的研究方法在定义包膜的单个组成部分方面非常重要，但在确定途径之间的联系方面受到限制，并且根据定义，低通量。我们的实验室（UCSF的Carol Gross实验室）之前采用了一种系统的方法来研究革兰氏阴性细菌大肠杆菌的包膜功能，方法是将一个基因缺失文库暴露在一组针对包膜的环境应激源中。利用这个数据集，我们能够在我们对肽聚糖（PG）合成的理解方面取得重大进展，肽聚糖是细菌细胞壁的关键成分。然而，革兰氏阳性菌（如枯草芽孢杆菌）的包膜与大肠杆菌的包膜有着根本的不同。枯草芽孢杆菌的包膜有一个外膜（因此有一个明显的质周间隙），包括大肠杆菌中没有的磷壁酸聚合物，并含有一层比大肠杆菌厚几倍的PG。在这项研究中，我将采用我们实验室以前使用的高通量方法来研究枯草芽孢杆菌包膜。首先，我将系统地鉴定枯草芽孢杆菌中所有非必需基因的单缺失突变和双缺失子集的表型。然后，我将使用这些数据集来研究特定的包膜过程，如调节ECF sigma因子的蛋白水解级联，包膜相关双组分系统的特征不明确的靶标，青霉素结合蛋白之间的功能冗余，以及涉及C55去戊二烯醇磷酸载体脂质的途径。最后，我将使用我在构建枯草芽孢杆菌缺失文库期间发现的新表型来确定ylaN的功能，这是一个涉及细胞形状的基因。这些高通量方法将加速对枯草芽孢杆菌中大量未表征的假定包膜基因的表型和功能的分配。这项研究的发现可能会扩展到重要的革兰氏阳性人类病原体，以及肠道微生物群中的人类共生体。

DESCRIPTION (provided by applicant): The bacterial cell is encased by an envelope that is in direct and constant contact with the environment. Thus, the envelope is the site where environmental fluctuations (e.g., changes in osmolarity) are first sensed by the bacterium. Also, components of the envelope mediate communication among other bacteria as well as commensal or pathogenic interactions between bacteria and their human hosts. These interactions play critical roles in complex environments such as the human gut microbiome. Finally, many of the most successful antibiotics in widespread use target the envelope (e.g., penicillin and vancomycin), and the efficacy of antibiotics with cytoplasmic targets is often reduced by issues with envelope permeability. For these reasons, the envelope is an area of intense research focus. Research approaches that investigate a single gene product or pathway have been immensely important in defining individual components of the envelope, but are limited in determining connections between pathways and are, by definition, low-throughput. Our lab (the Carol Gross lab at UCSF) previously took a systematic approach to investigate envelope function in the Gram-negative bacterium Escherichia coli by exposing a gene deletion library to a panel of environmental stressors that targeted the envelope. Using this dataset, we were able to make significant advances in our understanding of peptidoglycan (PG) synthesis, a key component of the bacterial cell wall. However, the envelope of Gram-positive bacteria, such as Bacillus subtilis, is fundamentally different from that of E. coli. The B. subtilis envelope lacs an outer membrane (and, thus, a distinct periplasmic space), includes teichoic acid polymers that are absent in E. coli, and contains a layer of PG that is several times thicker than found in E. coli. In this study, I will adapt the high-throughput approach previously used by our lab to investigate the B. subtilis envelope. First, I will systematically identify phenotypes for single deletion mutants of all non-essential genes in B. subtilis and a subset of double deletions. Then, I will use these datasets to investigate specific envelope processes such as proteolytic cascades that regulate ECF sigma factors, poorly characterized targets of the envelope-related two-component systems, functional redundancy amongst penicillin-binding proteins, and pathways involving the C55 undecaprenol phosphate carrier lipid. Finally, I will use a novel phenotype I discovered during construction of the B. subtilis deletion library to determine the function of ylaN, a gene involved in cell shape. These high-throughput methodologies will accelerate the assignment of phenotypes and functions to the large number of uncharacterized putative envelope genes in B. subtilis. The discoveries made in this study will likely extend to important Gram-positive human pathogens, as well as human commensals in the gut microbiome.