Horizontal gene transfer mediated by broad-host-range plasmids is an important mechanism of antibiotic resistance spread. While not all bacteria maintain plasmids equally well, plasmid persistence can improve over time, yet no general evolutionary mechanisms have emerged. Our goal was to identify these mechanisms, and to assess if adaptation to one plasmid affects the permissiveness to others. We experimentally evolved Pseudomonas sp. H2 containing multi-drug resistance plasmid RP4, determined plasmid persistence and cost using a joint experimental-modeling approach, resequenced evolved clones, and reconstructed key mutations. Plasmid persistence improved in fewer than 600 generations because the fitness cost turned into a benefit. Improved retention of naive plasmids indicated that the host evolved towards increased plasmid permissiveness. Key chromosomal mutations affected two accessory helicases and the RNA polymerase β-subunit. Our and other findings suggest that poor plasmid persistence can be caused by a high cost involving helicase-plasmid interactions that can be rapidly ameliorated.

The World Health Organization has declared the emergence of antibiotic resistance to be a global threat to human health. Broad-host-range plasmids have a key role in causing this health crisis because they transfer multiple resistance genes to a wide range of bacteria. To limit the spread of antibiotic resistance, we need to gain insight into the mechanisms by which the host range of plasmids evolves. Although initially unstable plasmids have been shown to improve their persistence through evolution of the plasmid, the host, or both, the means by which this occurs are poorly understood. Here, we sought to identify the underlying genetic basis of expanded plasmid host-range and increased persistence of an antibiotic resistance plasmid using a combined experimental-modeling approach that included whole-genome resequencing, molecular genetics and a plasmid population dynamics model. In nine of the ten previously evolved clones, changes in host and plasmid each slightly improved plasmid persistence, but their combination resulted in a much larger improvement, which indicated positive epistasis. The only genetic change in the plasmid was the acquisition of a transposable element from a plasmid native to the Pseudomonas host used in these studies. The analysis of genetic deletions showed that the critical genes on this transposon encode a putative toxin-antitoxin (TA) and a cointegrate resolution system. As evolved plasmids were able to persist longer in multiple naïve hosts, acquisition of this transposon also expanded the plasmid's host range, which has important implications for the spread of antibiotic resistance.

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SUMMARY Antibiotic selection drives adaptation of antibiotic resistance plasmids to new bacterial hosts, but the molecular mechanisms are still poorly understood. We previously showed that a broad-host-range plasmid was poorly maintained in Shewanella oneidensis, but rapidly adapted through mutations in the replication initiation gene trfA1. Here we examined if these mutations reduced the fitness cost of TrfA1, and whether this was due to changes in interaction with the host’s DNA helicase DnaB. The strains expressing evolved TrfA1 variants showed a higher growth rate than those expressing ancestral TrfA1. The evolved TrfA1 variants showed a lower affinity to the helicase than ancestral TrfA1 and were no longer able to activate the helicase at the oriV without host DnaA. Moreover, persistence of the ancestral plasmid was increased upon overexpression of DnaB. Finally, the evolved TrfA1 variants generated higher plasmid copy numbers than ancestral TrfA1. The findings suggest that ancestral plasmid instability can at least partly be explained by titration of DnaB by TrfA1. Thus under antibiotic selection resistance plasmids can adapt to a novel bacterial host through partial loss of function mutations that simultaneously increase plasmid copy number and decrease unfavorably high affinity to one of the hosts’ essential proteins.

ABSTRACT:Geobacillus thermoglucosidasius is a Gram-positive, thermophilic bacterium capable of ethanologenic fermentation of both C5 and C6 sugars and may have possible use for commercial bioethanol production [Tang et al., 2009;Taylor et al. (2009) Trends Biotechnol 27(7): 398-405]. Little is known about the physiological changes that accompany a switch from aerobic (high redox) to microaerobic/fermentative (low redox) conditions in thermophilic organisms. The changes in the central metabolic pathways in response to a switch in redox potential were analyzed using quantitative real-time PCR and proteomics. During low redox (fermentative) states, results indicated that glycolysis was uniformly up-regulated, the Krebs (tricarboxylic acid or TCA) cycle nonuniformly down-regulated and that there was little to no change in the pentose phosphate pathway. Acetate accumulation was accounted for by strong down-regulation of the acetate CoA ligase gene (acs) in addition to up-regulation of the pta and ackA genes (involved in acetate production), thus conserving ATP while reducing flux through the TCA cycle. Substitution of an NADH dehydrogenase (down-regulated) by an up-regulated NADH:FAD oxidoreductase and up-regulation of an ATP synthase subunit, alongside the observed shifts in the TCA cycle, suggested that an oxygen-scavenging electron transport chain likely remained active during low redox conditions. Together with the observed up-regulation of a glyoxalase and down-regulation of superoxide dismutase, thought to provide protection against the accumulation of toxic phosphorylated glycolytic intermediates and reactive oxygen species, respectively, the changes observed in G. thermoglucosidasius NCIMB 11955 under conditions of aerobic-to-microaerobic switching were consistent with responses to low pO 2 stress.