A trade-off for maintenance of multidrug-resistance IncHI2 plasmids in Salmonella Typhimurium through co-evolution. A trade-off for maintenance of multidrug-resistance IncHI2 plasmids in Salmonella Typhimurium through co-evolution

Plasmid maintenance costs to bacterial hosts is closely linked to the mechanisms that underlie plasmid fitness and how these costs are resolved. Herein, we performed multiple (63) serial passage to explore the compensatory mechanisms of co-evolution of multidrug-resistant IncHI2 plasmid pJXP9 and S. Typhimurium strain ATCC 14028 with or without antibiotic selection. pJXP9 could be maintained at for hundreds of generations even without drug exposure. Decreased lag times and higher competitive advantages were observed in end-point evolved strains bearing pJXP9 compared to ancestral strains. Genomic and transcriptomic analyses revealed that the fitness costs of pJXP9 in ATCC 14028 were derived from not only specific plasmid genes, particularly the multidrug-resistant region and conjugation transfer region I, but also the conflicts resulting from chromosomal gene interactions. Correspondingly, plasmid-borne deletions of these regions could compensate the fitness cost due to the presence of the plasmid. Furthermore, mutations and mRNA alterations in chromosomal genes involved in physiological functions were also adaptative. These functions included decreased flagellar motility, oxidative stress resistance and fumaric acid synthesis, and increased Cu resistance. Our findings suggest that plasmid maintenance through plasmid-bacteria co-evolution is a trade-off between increasing plasmid vertical transmission and impairing its horizontal transmission and bacterial physiological phenotypes. Overall design: RNA was extracted from clones cultured in LB broth after 4 h mating in the absence of antibiotics.Bacterial cells were collected by 8000×g centrifugation for 10 min. The library was constructed using an Illumina TruSeq RNA sample Prep Kit v2and sequenced by the Illumina Hiseq-2000. The reads that passed the Illumina quality filter were kept for sequence analysis.

质粒维持代价与细菌宿主的关联，核心在于质粒适合度背后的调控机制以及此类代价的缓解途径。本研究通过63次连续传代实验，探究了多重耐药IncHI2型质粒（IncHI2 plasmid）pJXP9与鼠伤寒沙门氏菌（S. Typhimurium）菌株ATCC 14028在有无抗生素筛选条件下的共进化补偿机制。结果显示，即使无药物暴露，pJXP9也可在宿主中稳定维持数百代。与亲本菌株相比，携带pJXP9的终点传代进化菌株表现出更短的延滞期与更强的竞争优势。基因组与转录组分析表明，ATCC 14028中pJXP9带来的适合度代价，不仅源自质粒上的特定基因（尤其是多重耐药区域与接合转移区域I），还源于染色体基因互作所引发的冲突。相应地，质粒上这些区域的缺失可有效缓解因质粒存在而产生的适合度代价。此外，参与生理功能的染色体基因所发生的突变与mRNA表达改变同样具有适应性，此类生理功能包括鞭毛运动性下降、抗氧化应激能力与富马酸合成的调控变化，以及铜抗性的增强。本研究结果提示，通过质粒-细菌共进化实现的质粒维持，本质是质粒垂直传播效率提升与质粒水平传播能力、细菌生理表型受损之间的一种进化权衡。整体实验设计：在无抗生素条件下进行4小时接合反应后，从LB肉汤中培养的克隆菌株中提取总RNA。以8000×g离心10分钟收集细菌细胞，使用Illumina TruSeq RNA样本制备试剂盒v2（Illumina TruSeq RNA Sample Prep Kit v2）构建测序文库，并通过Illumina Hiseq-2000测序平台（Illumina Hiseq-2000）完成测序。保留通过Illumina质量质控过滤的reads用于后续序列分析。