Luong Truc Thanh, Nguyen Minh Tan, Chen Yi-Wei, Chang Chungyu, Lee Ju Huck, Wittchen Manuel, Ton-That HyLam, Cruz Melissa, Garsin Danielle A, Das Asis, Tauch Andreas, Ton-That Hung
Division of Oral Biology & Medicine, School of Dentistry, University of California, Los Angeles, CA 90095, USA.
Department of Microbiology & Molecular Genetics, University of Texas Health Science Center, Houston, TX 77030, USA.
Microorganisms. 2021 Feb 14;9(2):389. doi: 10.3390/microorganisms9020389.
Controlled RNA degradation is a crucial process in bacterial cell biology for maintaining proper transcriptome homeostasis and adaptation to changing environments. mRNA turnover in many Gram-positive bacteria involves a specialized ribonuclease called RNase J (RnJ). To date, however, nothing is known about this process in the diphtheria-causative pathogen , nor is known the identity of this ribonuclease in this organism. Here, we report that DIP1463 encodes a predicted RnJ homolog, comprised of a conserved N-terminal β-lactamase domain, followed by β-CASP and C-terminal domains. A recombinant protein encompassing the β-lactamase domain alone displays 5'-exoribonuclease activity, which is abolished by alanine-substitution of the conserved catalytic residues His and His. Intriguingly, deletion of DIP1463/ in reduces bacterial growth and generates cell shape abnormality with markedly augmented cell width. Comparative RNA-seq analysis revealed that RnJ controls a large regulon encoding many factors predicted to be involved in biosynthesis, regulation, transport, and iron acquisition. One upregulated gene in the ∆ mutant is , coding for a membrane protease (FtsH) involved in cell division, whose overexpression in the wild-type strain also caused cell-width augmentation. Critically, the ∆ mutant is severely attenuated in virulence in a model of infection, while the FtsH-overexpressing and toxin-less strains exhibit full virulence as the wild-type strain. Evidently, RNase J is a key ribonuclease in that post-transcriptionally influences the expression of numerous factors vital to corynebacterial cell physiology and virulence. Our findings have significant implications for basic biological processes and mechanisms of corynebacterial pathogenesis.
可控的RNA降解是细菌细胞生物学中维持转录组稳态和适应环境变化的关键过程。许多革兰氏阳性菌中的mRNA周转涉及一种名为RNase J（RnJ）的特殊核糖核酸酶。然而，迄今为止，对于引起白喉的病原体中的这一过程以及该生物体中这种核糖核酸酶的身份仍一无所知。在此，我们报告DIP1463编码一种预测的RnJ同源物，它由一个保守的N端β-内酰胺酶结构域、随后的β-CASP和C端结构域组成。仅包含β-内酰胺酶结构域的重组蛋白表现出5'-外切核糖核酸酶活性，该活性会因保守催化残基His和His的丙氨酸取代而丧失。有趣的是，在中缺失DIP1463会降低细菌生长并导致细胞形状异常，细胞宽度显著增加。比较RNA-seq分析表明，RnJ控制着一个大的调控子，该调控子编码许多预测参与生物合成、调控、运输和铁获取的因子。∆突变体中一个上调的基因是，它编码一种参与细胞分裂的膜蛋白酶（FtsH），其在野生型菌株中的过表达也导致细胞宽度增加。至关重要的是，在感染模型中，∆突变体的毒力严重减弱，而FtsH过表达且无毒素的菌株表现出与野生型菌株相同的完全毒力。显然，RNase J是中的一种关键核糖核酸酶，它在转录后影响对棒状杆菌细胞生理学和毒力至关重要的众多因子的表达。我们的发现对棒状杆菌发病机制的基本生物学过程和机制具有重要意义。
