Alberts Maia E, Kurtz Micaila P, Müh Ute, Bernardi Jonathon P, Bollinger Kevin W, Dobrila Horia A, Duncan Leonard, Laster Hannah M, Orea Andres J, Pannullo Anthony G, Rivera-Rosado Juan G, Torres Facundo V, Ellermeier Craig D, Weiss David S
Department of Microbiology and Immunology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
Present address: Department of Anesthesiology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA.
bioRxiv. 2025 Jun 9:2025.06.04.657922. doi: 10.1101/2025.06.04.657922.
Essential genes are interesting in their own right and as potential antibiotic targets. To date, only one report has identified essential genes on a genome-wide scale in , a problematic pathogen for which treatment options are limited. That foundational study used large-scale transposon mutagenesis to identify 404 protein-encoding genes as likely to be essential for vegetative growth of the epidemic strain R20291. Here, we revisit the essential genes of strain R20291 using a combination of CRISPR interference (CRISPRi) and transposon-sequencing (Tn-seq). First, we targeted 181 of the 404 putatively essential genes with CRISPRi. We confirmed essentiality for >90% of the targeted genes and observed morphological defects for >80% of them. Second, we conducted a new Tn-seq analysis, which identified 346 genes as essential, of which 283 are in common with the previous report and might be considered a provisional essential gene set that minimizes false positives. We compare the list of essential genes to those of other bacteria, especially , highlighting some noteworthy differences. Finally, we used fusions to red fluorescent protein (RFP) to identify 18 putative new cell division proteins, three of which are conserved in Bacillota but of largely unknown function. Collectively, our findings provide new tools and insights that advance our understanding of .
必需基因本身就很有趣，并且作为潜在的抗生素靶点也很有意义。迄今为止，只有一份报告在全基因组范围内鉴定了一种治疗选择有限的致病性病原菌中的必需基因。该基础研究使用大规模转座子诱变来鉴定404个蛋白质编码基因可能是流行菌株R20291营养生长所必需的。在这里，我们结合CRISPR干扰（CRISPRi）和转座子测序（Tn-seq）重新审视R20291菌株的必需基因。首先，我们用CRISPRi靶向404个假定必需基因中的181个。我们确认了>90%的靶向基因的必需性，并观察到>80%的基因存在形态缺陷。其次，我们进行了一项新的Tn-seq分析，鉴定出346个基因为必需基因，其中283个与之前的报告相同，可能被视为一个将假阳性降至最低的临时必需基因集。我们将必需基因列表与其他细菌的列表进行比较，特别是，突出了一些值得注意的差异。最后，我们使用与红色荧光蛋白（RFP）的融合来鉴定18个假定的新细胞分裂蛋白，其中三个在芽孢杆菌门中保守，但功能大多未知。总的来说，我们的研究结果提供了新的工具和见解，推进了我们对的理解。
