Procuring Native Natural Product Producers by In Situ Chimera Assembly

描述（由申请人提供）：由于感染性疾病导致的死亡正在上升，主要是由于病原体耐药性的增加。因此，迫切需要开发新的抗生素来对抗抗微生物剂耐药性感染。从历史上看，细菌一直是新抗生素支架的最主要来源，证明了产生抗菌化合物的惊人能力。令人震惊的是，所有现有的基于天然产物的细菌衍生抗生素都来自地球微生物多样性的极小部分。具体来说，目前只有大约1%的细菌是可培养的，这意味着社会上所有最有价值的抗生素发现都来自于少数细菌分类群。宏基因组研究已经提出并证实，尚未培养的99%的细菌含有丰富的潜在抗生素物质等待发现。然而，目前从未培养的细菌中发掘这些化合物的方法带来了许多挑战，严重阻碍了有效开采这种资源的努力。考虑到这些局限性，该提案提出了一个新的假设，即尚未培养的细菌缺乏使可培养微生物能够在实验室中生存和增殖的基因和基因产物。为了验证这一假设，我们的团队正在开发一种创新技术，通过将来自实验室中表现出强劲生长的细菌的基因组DNA大片段引入尚未培养的细菌中，来创建可培养的嵌合克隆。这些拟议的研究将通过四个具体目标来完成。目的1涉及进行原位转染未培养的细菌在土壤和沉积物与细菌人工染色体（BAC）库组成的基因组DNA从一个容易培养的革兰氏阳性细菌和选择新的（以前未培养的）嵌合克隆。目的2旨在测试BAC编码基因的表达，鉴定基因插入物如何使生长成为可能，并评估微生物产生抗菌次级代谢产物。目的3将提供两个额外的BAC文库，其含有来自革兰氏阳性高GC细菌的基因组DNA和代表性的大部分未培养的但丰富的酸杆菌，用于进一步的嵌合体生成研究。目的4将测试这种方法从海洋无脊椎动物样品中产生嵌合克隆的能力，用于有针对性地采购两类抗感染天然产物的未培养微生物生产者。预计这项研究将产生若干重要成果。重要的是，我们将为使用这种创新技术培养以前未培养的细菌并获得其生物活性天然产物奠定基础。此外，我们预计一些新的化合物将包括现有可培养细菌中没有遇到的不寻常的支架。从长远来看，我们预计，来自以前未培养的细菌的新天然产物将提供大量机会，以获得迫切需要的新型抗菌剂，以控制因耐药性感染而造成的人类生命损失。

DESCRIPTION (provided by applicant): Deaths due to infectious diseases are on the rise fueled largely by an increase in pathogen drug resistance. Accordingly, there is a critical need for the development of new antibiotics to combat antimicrobial-resistant infections. Historically, bacteria have served as the foremost source of new antibiotic scaffolds demonstrating an incredible capacity for generating antimicrobial compounds. Shockingly, all existing natural-product-based bacterial-derived antibiotics have come from an extremely minor fraction of the Earth's microbial diversity. Specifically, only about 1% of bacteria are currently culturable meaning that all of society's most valuable antibiotic discoveries have originated from a decided minority of bacterial taxa. It has been proposed and substantiated by metagenome studies that the yet uncultured 99% of bacteria harbor an abundance of potential antibiotic substances awaiting discovery. However, current methods for unearthing these compounds from uncultured bacteria pose numerous challenges that severely hamper efforts to effectively mine this resource. Considering these limitations, this proposal poses the novel hypothesis that yet uncultured bacteria lack the genes and gene products that enable culturable microbes to survive and proliferate in the lab. To test this hypothesis, our group is developing an innovative technology for creating culturable chimeric clones by introducing large fragments of genomic DNA from bacteria exhibiting robust growth in the lab into as of yet uncultured bacteria. These proposed studies will be accomplished by means of four specific aims. Aim1 involves carrying out the in situ transfection of uncultured bacteria in soils and sediments with a bacterial artifical chromosome (BAC) library composed of genomic DNA from a readily culturable Gram-positive bacterium and selecting for new (previously uncultured) chimeric clones. Aim 2 is designed to test for the expression of BAC encoded genes, identify how the gene inserts enable growth, and assess microbes for the production of antimicrobial secondary metabolites. Aim 3 will provide two additional BAC libraries containing genomic DNA from a Gram-positive high-GC bacterium and a representative of the largely uncultured, but abundant Acidobacteria for further chimera-generation studies. Aim 4 will test the capacity of this method to generate chimeric clones from marine invertebrate samples for the targeted procurement of uncultured microbial producers of two classes of antiinfective natural products. Several important outcomes are anticipated as a result of this research. Importantly, we will establish a foundation for using this innovative technique to grow previously uncultured bacteria and obtain their bioactive natural products. In addition, we expect that some of the newly accessible compounds will include unusual scaffolds not encountered from existing culturable bacteria. In the long-term, we anticipate that new natural products from previously uncultured bacteria will provide abundant opportunities to procure novel antimicrobials that are desperately needed to control the increasing loss of human life attributable to antibiotic-resistant infections.