Computational protocol: Multidrug-resistant bacteria compensate for the epistasis between resistances

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Protocol publication

[…] The DNA extracted was sequenced using the Miseq Illumina platform. Coverage of the different pools was as follows: 521x for 12 clones in the RifR pool, 640x for 12 clones in the StrR pool, and 631x for 24 clones in the RifR StrR pool. The reads were filtered using SeqTk version 1.0-r63. The resulting sequences were analyzed in Breseq version 2.3, using E. coli K12 genome NC_000913.2 as a reference, and with the polymorphism option selected and the following parameters: (a) rejection of polymorphisms in homopolymers of a length greater than three, (b) rejection of polymorphisms that are not present in at least three reads in each strand, and (c) rejection of polymorphisms that do not have a p-value for quality greater than 0.05. All other Breseq parameters were used as default. Mutations in homopolymers and pseudogenes were also discarded. The genomic data in was plotted using the BLAST Ring Image Generator (BRIG) software []. [...] The mutations located in nusE and nusG regulatory regions were constructed by Lambda-Red recombineering [] using a resistance cassette inserted in a nearby gene (gspB and tufB, respectively) as a marker for selection, followed by transference to RifR, StrR, and RifR StrR backgrounds by P1 transduction [] using the same marker. Derivatives of these strains harboring the marker in the nearby gene but not the mutations in nusE and nusG were constructed, in order to be used as references in the competition experiments. The putative compensatory mutations located in rpoC were constructed by pORTMAGE recombineering [] in both the RifR and the StrR backgrounds and subsequent transfer to the sensitive, RifR, StrR, and RifR StrR backgrounds was done by P1 transduction, using as recipient auxotroph derivatives carrying a mutation in the nearby gene argE and selecting for growth in minimal medium. The primers used are listed in the . The presence of the desired mutations in the transductant isolates was assessed by PCR-mediated amplification of the corresponding gene and sequencing. Both reconstructed strains and the corresponding reference strains were unfrozen and acclimatized during 24 h to avoid compensatory mutations appearing in the more costly backgrounds. Reconstructed mutants were competed against the respective ancestral strain to assess their competitive advantage per generation, assuming the competition lasts eight generations, or 24 h. The fitness landscapes of the main reconstructed compensatory genotypes () were plotted using the MAGELLAN (Maps of Genetical Landscapes) software []. […]

Pipeline specifications

Software tools Seqtk, breseq, BRIG, MAGELLAN
Applications Genome annotation, WGS analysis, Genome data visualization
Organisms Escherichia coli, Bacteria
Chemicals Rifampin, Streptomycin