Plasmids play an important role in bacterial genome evolution by transferring genes between lineages. Fitness costs associated with plasmid carriage are expected to be a barrier to gene exchange, but the causes of plasmid fitness costs are poorly understood. Single compensatory mutations are often sufficient to completely ameliorate plasmid fitness costs, suggesting that such costs are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or gene expression level. By combining the results of experimental evolution with genetics and transcriptomics, we show here that fitness costs of 2 divergent large plasmids in Pseudomonas fluorescens are caused by inducing maladaptive expression of a chromosomal tailocin toxin operon. Mutations in single genes unrelated to the toxin operon, and located on either the chromosome or the plasmid, ameliorated the disruption associated with plasmid carriage. We identify one of these compensatory loci, the chromosomal gene PFLU4242, as the key mediator of the fitness costs of both plasmids, with the other compensatory loci either reducing expression of this gene or mitigating its deleterious effects by up-regulating a putative plasmid-borne ParAB operon. The chromosomal mobile genetic element Tn6291, which uses plasmids for transmission, remained up-regulated even in compensated strains, suggesting that mobile genetic elements communicate through pathways independent of general physiological disruption. Plasmid fitness costs caused by specific genetic conflicts are unlikely to act as a long-term barrier to horizontal gene transfer (HGT) due to their propensity for amelioration by single compensatory mutations, helping to explain why plasmids are so common in bacterial genomes.

Plasmids play an important role in bacterial genome evolution by transferring genes between lineages. Fitness costs associated with plasmid acquisition are expected to be a barrier to gene exchange, but the causes of plasmid fitness costs are poorly understood. Single compensatory mutations are often sufficient to completely ameliorate plasmid fitness costs, suggesting that such costs are caused by specific genetic conflicts rather than generic properties of plasmids, such as their size, metabolic burden, or expression level. Here we show -- using a combination of experimental evolution, reverse genetics, and transcriptomics -- that fitness costs of two divergent large plasmids in Pseudomonas fluorescens are caused by inducing maladaptive expression of a chromosomal tailocin toxin operon. Mutations in single genes unrelated to the toxin operon, and located on either the chromosome or the plasmid, ameliorated the disruption associated with plasmid acquisition. We identify one of these compensatory loci, the chromosomal gene PFLU4242, as the key mediator of the fitness costs of both plasmids, with the other compensatory loci either reducing expression of this gene or mitigating its deleterious effects by upregulating a putative plasmid-borne ParAB operon. The chromosomal mobile genetic element Tn6291, which uses plasmids for transmission, remained upregulated even in compensated strains, suggesting that mobile genetic elements communicate through pathways independent of general physiological disruption. Plasmid fitness costs caused by specific genetic conflicts are unlikely to act as a long-term barrier to horizontal gene transfer due to their propensity for amelioration by single compensatory mutations, explaining why plasmids are so common in bacterial genomes.

Introduction. Cupriavidus pauculus is historically found in soil and water but has more recently been reported to cause human infection and death. Hospital sink traps can serve as a niche for bacterial persistence and a platform for horizontal gene transfer, with evidence of dissemination of pathogens in hospital plumbing systems driving nosocomial infection. Gap Statement. This paper presents the first C. pauculus strain isolated from a hospital sink trap. There are only six genome assemblies available on NCBI for C. pauculus ; two of these are PacBio/Illumina hybrids. This paper presents the first ONT/Illumina hybrid assembly, with five contigs. The other assemblies available consist of 37, 38, 111 and 227 contigs. This paper also presents data on biofilm formation and lethal dose in Galleria mellonella; there is little published information describing these aspects of virulence. Aim. The aims were to identify the isolate found in a hospital sink trap, characterize its genome, and assess whether it could pose a risk to human health. Methodology. The genome was sequenced, and a hybrid assembly of short and long reads produced. Antimicrobial susceptibility was determined by the broth microdilution method. Virulence was assessed by measuring in vitro biofilm formation compared to Pseudomonas aeruginosa and in vivo lethality in Galleria mellonella larvae. Results. The isolate was confirmed to be a strain of C. pauculus , with a 6.8 Mb genome consisting of 6468 coding sequences and an overall G+C content of 63.9 mol%. The genome was found to contain 12 antibiotic resistance genes, 8 virulence factor genes and 33 metal resistance genes. The isolate can be categorized as resistant to meropenem, amoxicillin, amikacin, gentamicin and colistin, but susceptible to cefotaxime, cefepime, imipenem and ciprofloxacin. Clear biofilm formation was seen in all conditions over 72 h and exceeded that of P. aeruginosa when measured at 37 °C in R2A broth. Lethality in G. mellonella larvae over 48 h was relatively low. Conclusion. The appearance of a multidrug-resistant strain of C. pauculus in a known pathogen reservoir within a clinical setting should be considered concerning. Further work should be completed to compare biofilm formation and in vivo virulence between clinical and environmental strains, to determine how easily environmental strains may establish human infection. Infection control teams and clinicians should be aware of the emerging nature of this pathogen and further work is needed to minimize the impact of contaminated hospital plumbing systems on patient outcomes.