Institute of Technology, University of Tartu, 50411, Tartu, Estonia.
Institute of Pharmacy, University of Tartu, 50411, Tartu, Estonia.
Sci Rep. 2024 Mar 22;14(1):6883. doi: 10.1038/s41598-024-57597-8.
We developed a reporter system based on simultaneous expression of two fluorescent proteins: GFP as a reporter of the capacity of protein synthesis and mutated mScarlet-I as a reporter of translational errors. Because of the unique stop codons or frameshift mutations introduced into the mScarlet-I gene, red fluorescence was produced only after a mistranslation event. These reporters allowed us to estimate mistranslation at a single cell level using either flow cytometry or fluorescence microscopy. We found that laboratory strains of Escherichia coli are more prone to mistranslation compared to the clinical isolates. As relevant for uropathogenic E. coli, growth in human urine elevated translational frameshifting compared to standard laboratory media, whereas different standard media had a small effect on translational fidelity. Antibiotic-induced mistranslation was studied by using amikacin (aminoglycoside family) and azithromycin (macrolide family). Bactericidal amikacin induced preferably stop-codon readthrough at a moderate level. Bacteriostatic azithromycin on the other hand induced both frameshifting and stop-codon readthrough at much higher level. Single cell analysis revealed that fluorescent reporter-protein signal can be lost due to leakage from a fraction of bacteria in the presence of antibiotics, demonstrating the complexity of the antimicrobial activity.
GFP 作为蛋白质合成能力的报告者，以及突变的 mScarlet-I 作为翻译错误的报告者。由于 mScarlet-I 基因中引入了独特的终止密码子或移码突变，只有在发生翻译错误后才会产生红色荧光。这些报告者允许我们使用流式细胞术或荧光显微镜在单细胞水平上估计翻译错误。我们发现，与临床分离株相比，实验室菌株的大肠杆菌更容易发生翻译错误。对于尿路致病性大肠杆菌，与标准实验室培养基相比，在人尿中生长会增加翻译移码，而不同的标准培养基对翻译保真度的影响较小。通过使用阿米卡星（氨基糖苷类）和阿奇霉素（大环内酯类）研究了抗生素诱导的翻译错误。杀菌性阿米卡星以中等水平优先诱导终止密码子通读。另一方面，抑菌性阿奇霉素以更高的水平诱导移码和终止密码子通读。单细胞分析表明，在存在抗生素的情况下，由于一部分细菌的渗漏，荧光报告蛋白信号可能会丢失，这表明了抗生素活性的复杂性。
