Procuring Native Natural Product Producers by In Situ Chimera Assembly

描述（由申请人提供）：传染病造成的死亡人数正在上升，这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菌素耐药感染。从历史上看，细菌一直是新型抗生素支架的主要来源，显示出产生抗菌化合物的令人难以置信的能力。令人震惊的是，所有现有的基于天然产品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，这意味着所有社会上最有价值的抗生素发现都起源于少数细菌分类群。宏基因组研究已经提出并证实，尚未培养的99%细菌中含有大量潜在的抗生素物质，等待发现。然而，目前从未培养的细菌中挖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。考虑到这些限制，这一建议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，通过将实验室中生长强劲的细菌的大片段基因组DNA引入尚未培养的细菌中，来创造可培养的嵌合克隆。这些拟议的研究将通过四个具体目标来完成。目的1包括用细菌人工染色体（BAC）文库对土壤和沉积物中未培养的细菌进行原位转染，该文库由易于培养的革兰氏阳性细菌的基因组DNA组成，并选择新的（先前未培养的）嵌合克隆。目的2旨在检测BAC编码基因的表达，确定基因插入物如何促进生长，并评估微生物产生抗菌次级代谢物的能力。Aim 3将提供两个额外的BAC文库，其中包含来自革兰氏阳性高gc细菌的基因组DNA和大部分未培养但丰富的酸杆菌的代表，用于进一步的嵌合体代研究。目标4将测试该方法从海洋无脊椎动物样本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品的未培养微生物生产者。预计这项研究将产生几个重要的结果。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，我们预计一些新的可获得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，我们预计从以前未培养的细菌中获得的新的天然产物将为获得新型抗菌素提供大量机会，这些抗菌素是控制抗生素耐药感染造成的日益增加的人类生命损失所迫切需要的。

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.