A Novel Small RNA on the Pseudomonas putida KT2440 Chromosome Is Involved in the Fitness Cost Imposed by IncP-1 Plasmid RP4
A Novel Small RNA on the Pseudomonas putida KT2440 Chromosome Is Involved in the Fitness Cost Imposed by IncP-1 Plasmid RP4
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DOI：
10.3389/fmicb.2020.01328
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发表时间：
2020-06
影响因子：
5.2
通讯作者：
Hibiki Kawano;C. Suzuki-Minakuchi;D. Sugiyama;N. Watanabe;Yurika Takahashi;K. Okada;H. Nojiri
中科院分区：
文献类型：
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作者：
Hibiki Kawano;C. Suzuki-Minakuchi;D. Sugiyama;N. Watanabe;Yurika Takahashi;K. Okada;H. Nojiri
质粒可以为宿主细菌提供有利的特性，尽管它们可能会带来适应性成本。染色体编码因子对于调节质粒上基因的表达是重要的，宿主染色体可能在它们与给定质粒的相互作用方面有所不同。因此，不同的宿主染色体/质粒组合在适应成本负荷和代偿性协同进化方面可能存在差异。然而，代偿进化的机制是高度分化的，需要进一步的了解。本研究揭示了恶臭假单胞菌KT2440提高不相容P-1 （IncP-1）多药耐药质粒RP4适应度成本的新进化机制。在非选择性条件下，每24 h连续转移含rp4细胞和无KT2440细胞的混合培养。最初，含rp4细胞的比例迅速下降，但很快恢复，说明在培养过程中，含rp4菌株的适应度提高了。混合培养144 h时出现了较大的菌落，从较大菌落分离出的进化菌株比祖先菌株具有更高的生长速率和适合度。全基因组测序显示，进化菌株在染色体的同一基因间区域有两个突变之一。根据另一组的研究，该区域被预测含有应激诱导的小RNA （sRNA）。对该sRNA转录起始位点的鉴定表明，一个突变发生在sRNA区域内，而另一个突变发生在其启动子区域。定量反转录PCR结果显示，该sRNA在祖先菌株中被携带RP4的菌株强烈诱导表达，而在进化菌株中被抑制。当sRNA区域在无rp4菌株中过表达时，适应度降低，菌落大小变小。通过转录组分析，我们还发现参与氨基酸代谢和应激反应的基因通过sRNA区域的过表达而差异转录。这些结果表明，r4诱导的染色体sRNA负责r4在KT2440细胞上的适应度成本，该sRNA在宿主进化中朝着快速改善成本的方向发挥关键作用。
Plasmids can provide advantageous traits to host bacteria, although they may impose a fitness cost. Chromosome-encoded factors are important for regulating the expression of genes on plasmids, and host chromosomes may differ in terms of their interactions with a given plasmid. Accordingly, differences in fitness cost loading and compensatory co-evolution may occur for various host chromosome/plasmid combinations. However, the mechanisms of compensatory evolution are highly divergent and require further insights. Here, we reveal novel evolutionally mechanisms of Pseudomonas putida KT2440 to improve the fitness cost imposed by the incompatibility P-1 (IncP-1) multidrug resistance plasmid RP4. A mixed culture of RP4-harboring and -free KT2440 cells was serially transferred every 24 h under non-selective conditions. Initially, the proportion of RP4-harboring cells decreased rapidly, but it immediately recovered, suggesting that the fitness of RP4-harboring strains improved during cultivation. Larger-sized colonies appeared during 144-h mixed culture, and evolved strains isolated from larger-sized colonies showed higher growth rates and fitness than those of the ancestral strain. Whole-genome sequencing revealed that evolved strains had one of two mutations in the same intergenic region of the chromosome. Based on the research of another group, this region is predicted to contain a stress-inducible small RNA (sRNA). Identification of the transcriptional start site in this sRNA indicated that one mutation occurred within the sRNA region, whereas the other was in its promoter region. Quantitative reverse-transcription PCR showed that the expression of this sRNA was strongly induced by RP4 carriage in the ancestral strain but repressed in the evolved strains. When the sRNA region was overexpressed in the RP4-free strain, the fitness decreased, and the colony size became smaller. Using transcriptome analysis, we also showed that the genes involved in amino acid metabolism and stress responses were differentially transcribed by overexpression of the sRNA region. These results indicate that the RP4-inducible chromosomal sRNA was responsible for the fitness cost of RP4 on KT2440 cells, where this sRNA is of key importance in host evolution toward rapid amelioration of the cost.