Evolved plasmid-host interactions reduce plasmid interference cost.

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• Antibiotic resistance plasmids evolve to new bacterial hosts, but mechanisms remain unclear.
• Previous work showed rapid adaptation of a plasmid in Shewanella oneidensis via mutations in trfA1.
• The fitness cost and molecular basis of this adaptation were not fully understood.

Purpose of the Study:

• Investigate if trfA1 mutations reduce plasmid fitness cost in S. oneidensis.
• Determine if adaptation involves altered interactions between TrfA1 and host DNA helicase DnaB.
• Elucidate the impact of TrfA1 evolution on plasmid stability and copy number.

Main Methods:

• Compared growth rates of S. oneidensis strains with ancestral and evolved TrfA1 variants.
• Assessed TrfA1-DnaB binding affinity using biochemical assays.
• Examined helicase activation at the origin of replication (oriV) with and without host DnaA.
• Investigated plasmid persistence upon DnaB overexpression.
• Quantified plasmid copy number for ancestral and evolved variants.

Main Results:

• Evolved TrfA1 variants exhibited higher growth rates than ancestral TrfA1.
• Evolved TrfA1 showed reduced affinity for DnaB and required host DnaA for helicase activation at oriV.
• Overexpression of DnaB increased persistence of the ancestral plasmid.
• Evolved TrfA1 variants resulted in higher plasmid copy numbers.

Conclusions:

• Plasmid instability in S. oneidensis is partly due to DnaB titration by ancestral TrfA1.
• Adaptation involves mutations conferring partial loss of TrfA1 function.
• These mutations increase plasmid copy number and decrease affinity for essential host proteins, facilitating adaptation under antibiotic selection.