Abstract:
Toxin-antitoxin systems are preserved by nearly every prokaryote. The type II toxin MazF acts as a sequence-specific endoribonuclease, cleaving ribonucleotides at specific sequences that vary from three to seven bases, as has been reported in different host organisms to date. The present study characterized the MazEF module (MazEF-sth) conserved in the Symbiobacterium thermophilum IAM14863 strain, a Gram-negative syntrophic bacterium that can be supported by co-culture with multiple bacteria, including Bacillus subtilis. Based on a method combining massive parallel sequencing and the fluorometric assay, MazF-sth was determined to cleave ribonucleotides at the UACAUA motif, which is markedly similar to the motifs recognized by MazF from B. subtilis (MazF-bs), and by several MazFs from Gram-positive bacteria. MazF-sth, with mutations at conserved amino acid residues Arg29 and Thr52, lost most ribonuclease activity, indicating that these residues that are crucial for MazF-bs also play significant roles in MazF-sth catalysis. Further, cross-neutralization between MazF-sth and the non-cognate MazE-bs was discovered, and herein, the neutralization mechanism is discussed based on a protein-structure simulation via AlphaFold2 and multiple sequence alignment. The conflict between the high homology shared by these MazF amino acid sequences and the few genetic correlations among their host organisms may provide evidence of horizontal gene transfer.
摘要:
几乎所有原核生物都保留了毒素-抗毒素系统。II型毒素MazF充当序列特异性核糖核酸内切酶,在3至7个碱基的特定序列上切割核糖核苷酸,正如迄今为止在不同宿主生物中所报道的那样。本研究表征了共生嗜热杆菌IAM14863菌株中保存的MazEF模块(MazEF-sth),可以通过与多种细菌共培养来支持的革兰氏阴性同养细菌,包括枯草芽孢杆菌.基于大规模平行测序和荧光分析相结合的方法,MazF-sth被确定在UACAUA基序切割核糖核苷酸,这与MazF从B.subtilis(MazF-bs)识别的图案明显相似,以及来自革兰氏阳性细菌的几种MazF。MazF-sth,在保守的氨基酸残基Arg29和Thr52突变,失去了大部分核糖核酸酶活性,表明这些对MazF-bs至关重要的残基在MazF-sth催化中也起着重要作用。Further,发现了MazF-sth和非同源MazE-bs之间的交叉中和,在这里,基于通过AlphaFold2和多序列比对的蛋白质结构模拟,讨论了中和机制。这些MazF氨基酸序列共有的高同源性与其宿主生物之间的少数遗传相关性之间的冲突可能提供水平基因转移的证据。