Plasmid fitness costs are caused by specific genetic conflicts enabling resolution by compensatory mutation

Abstract

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.

Translational demand is not a major source of plasmid-associated fitness costs

“…GFP, LacZ), and the fitness effects detected were only apparent when the overexpressed protein comprised a sizable fraction of the total protein pool of the cell (typically above 5%) [ 15 , 20 , 21 , 36 ]. This might be a key difference with our approximation, because despite the relatively high level of expression of plasmid-borne genes [ 37 , 38 ], the overall proportion of plasmid transcripts is relatively low (1–3% of total mRNA levels) [ 37 , 39 ]. If we assume protein levels to be roughly proportional to mRNA levels [ 40 ], this result would indicate that plasmid proteins comprise a relatively small fraction of the total protein cellular content, and that translation of plasmid genes is low enough to not significantly disturb the translational machinery of the cell.…”

“…Second, the CAI (a measure of codon usage similarity [ 23 ]) of plasmid genes weighted by their expression levels does not correlate with plasmid fitness costs [ 37 ]. And third, several studies have mechanistically explored the basis of plasmid costs [ 37 , 38 , 42 – 45 ], with only one pointing out a causal relationship between translation and plasmid costs [ 42 ]. Instead, the key insight that emerges from these studies is that plasmid costs are mainly caused by genomic and metabolic conflicts arising between plasmid and chromosomal genes [ 37 , 38 , 43 – 45 ].…”

“…R. Soc. B 377: 20200463 genomic and metabolic conflicts arising between plasmid and chromosomal genes [37,38,[43][44][45].…”

Mathematical Models of Plasmid Population Dynamics

“…Another important feature of mobile genetic elements (MGEs) is that they can impose a physiological burden on the host in the absence of selection for the traits they encode ( San Millan and MacLean, 2017 ). There are multiple mechanisms that have been shown experimentally to produce a fitness reduction to the host, for instance the energetic cost associated with replicating additional DNA ( Glick, 1995 ) or increasing protein synthesis ( Rozkov et al, 2004 ), as well as the production of plasmid gene products that induce genetic conflicts between plasmids and their hosts ( Hall et al, 2021 ) or that inhibit cell division until sufficient plasmid copies are available ( Nordström, 1985 ).…”

Introduction: the secret lives of microbial mobile genetic elements

“…It is not clear whether or how the wild-type version of skaA benefits the plasmid, but the gene appears to be essential for plasmid replication. Regardless, the fact that single basepair mutations can have such dramatic effects on costly plasmid-conferred phenotypes speaks to the importance of specific gene functions, and gene interactions within the context of a host cell [ 18 , 30 ]. With large MGEs carrying huge numbers of genes of unknown function and activity, there is great potential for conflicts to arise, and likewise for new adaptive phenotypes to emerge.…”