Michael G. DeGroote Institute for Infectious Disease Research, Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario, Canada.
Bioinformatics Research Group, SRI International, Menlo Park, California, USA.
mBio. 2020 Sep 29;11(5):e02259-20. doi: 10.1128/mBio.02259-20.
Central metabolism is a topic that has been studied for decades, and yet, this process is still not fully understood in , perhaps the most amenable and well-studied model organism in biology. To further our understanding, we used a high-throughput method to measure the growth kinetics of each of 3,796 single-gene deletion mutants in 30 different carbon sources. In total, there were 342 genes (9.01%) encompassing a breadth of biological functions that showed a growth phenotype on at least 1 carbon source, demonstrating that carbon metabolism is closely linked to a large number of processes in the cell. We identified 74 genes that showed low growth in 90% of conditions, defining a set of genes which are essential in nutrient-limited media, regardless of the carbon source. The data are compiled into a Web application, Carbon Phenotype Explorer (CarPE), to facilitate easy visualization of growth curves for each mutant strain in each carbon source. Our experimental data matched closely with the predictions from the EcoCyc metabolic model which uses flux balance analysis to predict growth phenotypes. From our comparisons to the model, we found that, unexpectedly, phosphoenolpyruvate carboxylase () was required for robust growth in most carbon sources other than most trichloroacetic acid (TCA) cycle intermediates. We also identified 51 poorly annotated genes that showed a low growth phenotype in at least 1 carbon source, which allowed us to form hypotheses about the functions of these genes. From this list, we further characterized the gene and demonstrated its role in adenosine efflux. While there has been much study of bacterial gene dispensability, there is a lack of comprehensive genome-scale examinations of the impact of gene deletion on growth in different carbon sources. In this context, a lot can be learned from such experiments in the model microbe where much is already understood and there are existing tools for the investigation of carbon metabolism and physiology (1). Gene deletion studies have practical potential in the field of antibiotic drug discovery where there is emerging interest in bacterial central metabolism as a target for new antibiotics (2). Furthermore, some carbon utilization pathways have been shown to be critical for initiating and maintaining infection for certain pathogens and sites of infection (3-5). Here, with the use of high-throughput solid medium phenotyping methods, we have generated kinetic growth measurements for 3,796 genes under 30 different carbon source conditions. This data set provides a foundation for research that will improve our understanding of genes with unknown function, aid in predicting potential antibiotic targets, validate and advance metabolic models, and help to develop our understanding of metabolism.
中心代谢是一个已经研究了几十年的课题，但在生物学中最易于研究和研究最充分的模式生物中，这个过程仍然没有被完全理解。为了进一步了解这一过程，我们使用高通量方法测量了 30 种不同碳源中 3796 个单基因缺失突变体的生长动力学。总共有 342 个基因（9.01%）涵盖了广泛的生物学功能，这些基因在至少一种碳源上表现出生长表型，这表明碳代谢与细胞内的许多过程密切相关。我们鉴定出 74 个基因在 90%的条件下生长缓慢，这一定程度上定义了一组在营养受限的培养基中必不可少的基因，而不论碳源如何。这些数据被汇编成一个网络应用程序，即碳表型浏览器（CarPE），以方便轻松地可视化每个突变菌株在每种碳源下的生长曲线。我们的实验数据与 EcoCyc 代谢模型的预测非常吻合，该模型使用通量平衡分析来预测生长表型。从我们与该模型的比较中，我们发现出乎意料的是，磷酸烯醇丙酮酸羧化酶（）对于大多数除三羧酸（TCA）循环中间体外的碳源的稳健生长是必需的。我们还鉴定出 51 个注释较差的基因，它们在至少一种碳源中表现出生长缓慢的表型，这使我们能够对这些基因的功能形成假设。从这个列表中，我们进一步表征了 基因，并证明了它在腺苷外排中的作用。尽管已经对细菌基因的非必需性进行了大量研究，但在不同碳源中基因缺失对生长的影响缺乏全面的全基因组研究。在这种情况下，可以从模型微生物的此类实验中吸取很多经验，因为在模型微生物中已经了解了很多，并且有用于研究碳代谢和生理学的现有工具（1）。基因缺失研究在抗生素药物发现领域具有实际潜力，因为人们对细菌中心代谢作为新抗生素的靶点越来越感兴趣（2）。此外，某些碳利用途径已被证明对某些病原体和感染部位的感染起始和维持至关重要（3-5）。在这里，我们使用高通量固体培养基表型方法，在 30 种不同碳源条件下为 3796 个基因生成了动力学生长测量值。该数据集为研究提供了基础，将有助于提高对未知功能基因的理解，有助于预测潜在的抗生素靶标，验证和推进代谢模型，并帮助我们更好地了解 代谢。
