A Novel Small RNA on thePseudomonas putidaKT2440 Chromosome Is Involved in the Fitness Cost Imposed by IncP-1 Plasmid RP4

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 ofPseudomonas putidaKT2440 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.