Department of Biological Sciences, Arkansas State University, Jonesboro, AR, USA.
Department of Oral Biology, University of Florida, Gainesville, FL, USA.
Microbiology (Reading). 2022 Oct;168(10). doi: 10.1099/mic.0.001254.
Integrative and conjugative elements (ICEs) are chromosomally encoded mobile genetic elements that can transfer DNA between bacterial strains. Recently, as part of efforts to determine hypothetical gene functions, we have discovered an important regulatory module encoded on an ICE known as TnSmu1 on the chromosome. The regulatory module consists of a cI-like repressor with a helix-turn-helix DNA binding domain (immunity repressor) and a metalloprotease (anti-repressor). It is not possible to create an in-frame deletion mutant of and repression of with CRISPRi (CRISPR interference) causes substantial cell defects. We used a bypass of essentiality (BoE) screen to discover genes that allow deletion of the regulatory module. This revealed that conjugation genes, located within TnSmu1, can restore the viability of an mutant. Deletion of also leads to production of a circular intermediate form of TnSmu1, which is also inducible by the genotoxic agent mitomycin C. To gain further insights into potential regulation of TnSmu1 by ImmR and broader effects on UA159 physiology, we used CRISPRi and RNA-seq. Strongly induced genes included all the TnSmu1 mobile element, genes involved in amino acid metabolism, transport systems and a type I-C CRISPR-Cas system. Lastly, bioinformatic analysis shows that the TnSmu1 mobile element and its associated genes are well distributed across isolates. Taken together, our results show that activation of TnSmu1 is controlled by the module, and that activation is deleterious to , highlighting the complex interplay between mobile elements and their host.
整合子-接合元件（ICEs）是染色体编码的可移动遗传元件，可在细菌菌株之间转移 DNA。最近，作为确定假设基因功能的努力的一部分，我们在染色体上发现了一个重要的调节模块，该模块编码在一个称为 TnSmu1 的 ICE 上。该调节模块由一个 cI 样抑制剂和一个金属蛋白酶（反抑制剂）组成。由于 CRISPRi（CRISPR 干扰）不可能创建一个框内缺失突变体，因此抑制 repressor 会导致细胞严重缺陷。我们使用必需性旁路（BoE）筛选来发现允许删除调节模块的基因。这表明位于 TnSmu1 内的接合基因可以恢复 repressor 突变体的活力。缺失 repressor 也会导致 TnSmu1 的圆形中间形式的产生，该形式也可被遗传毒性剂丝裂霉素 C 诱导。为了进一步了解 ImmR 对 TnSmu1 的潜在调节作用以及对 UA159 生理学的更广泛影响，我们使用了 CRISPRi 和 RNA-seq。强烈诱导的基因包括 TnSmu1 移动元件的所有基因、参与氨基酸代谢、运输系统和 I-C 型 CRISPR-Cas 系统的基因。最后，生物信息学分析表明，TnSmu1 移动元件及其相关基因在 分离株中分布良好。总之，我们的结果表明，TnSmu1 的激活受 模块控制，激活对 是有害的，这突出了移动元件与其宿主之间的复杂相互作用。
