We constructed double-resistant genotypes, with two resistance mutations or one resistance mutation plus the plasmid, by transduction or transformation as described below.
Consistent with previous studies [ 3, 5, 6, 15, 35], we found pairwise epistatic interactions among resistance elements on the same genetic background: out of seven multiple-resistant genotypes with a RifR mutation and either an StrR mutation or the plasmid, three showed significant deviations from a multiplicative model of fitness (Additional file 2: Table S2).
Crucially, this adaptation is as likely to be due to mutations on the host's chromosome as it is a mutation on the plasmid itself (Bouma and Lenski 1988; Dahlberg and Chao 2003; Dionisio et al. 2005).
Additionally, we tested pairwise epistatic interactions of RifR mutations with StrR mutations or the plasmid in genotypes that had not adapted to LB.
To eliminate the effect of mutation in the plasmid, we performed plasmid curing of the resistant mutant 1F2 and then pBBR1 C1PsABRe plasmid was transformed again.
Alternatively, it is possible that the mutation in the plasmid affects correct partitioning and increases instability.
Strains of Yersinia used included a pgm-, pPst- cured mutant of the C092 strain of Y. pestis (CO92 pPst− pgm−) and Y. pseudotuberculosis strains YpIII p(IB19), which has an in-frame deletion mutation of the plasmid pLcr-encoded lcrV gene, and strain Y. ptb.
However, they did identify a mutation within the plasmid which led to a further enhancement in the violacein yields [ 55].
To test for a CHXR phenotype caused by mutation of the plasmid borne CYH2 gene, isolates were replica-plated to synthetic dextrose medium lacking tryptophan (SD-Trp).
The non-suppressor strain W3101 was transformed by pD and pD::eGFP given that the ability to properly package and produce viable λ Dam15 particles would rely solely upon in trans complementation for the Dam15 mutation from the plasmid.
