Chromosomal barcoding as a tool for multiplexed phenotypic characterization of laboratory evolved lineages

Abstract

Adaptive laboratory evolution is an important tool to evolve organisms to increased tolerance towards different physical and chemical stress. It is applied to study the evolution of antibiotic resistance as well as genetic mechanisms underlying improvements in production strains. Adaptive evolution experiments can be automated in a high-throughput fashion. However, the characterization of the resulting lineages can become a time consuming task, when the performance of each lineage is evaluated individually. Here, we present a novel method for the markerless insertion of randomized genetic barcodes into the genome of Escherichia coli using a novel dual-auxotrophic selection approach. The barcoded E. coli library allows multiplexed phenotyping of evolved strains in pooled competition experiments. We use the barcoded library in an adaptive evolution experiment; evolving resistance towards three common antibiotics. Comparing this multiplexed phenotyping with conventional susceptibility testing and growth-rate measurements we can show a significant positive correlation between the two approaches. Use of barcoded bacterial strain libraries for individual adaptive evolution experiments drastically reduces the workload of characterizing the resulting phenotypes and enables prioritization of lineages for in-depth characterization. In addition, barcoded clones open up new ways to profile community dynamics or to track lineages in vivo or situ.

Dominant resistance and negative epistasis can limit the co-selection of de novo resistance mutations and antibiotic resistance genes

“…This could be explained by changes in fitness in the presence of the antibiotic or by population dynamics between different mutants in the mutant pool. Unexpected selection and cross resistance between unrelated conditions has been previously reported for Cefepime, highlighting previous adaptations and population dynamics can impact selection patterns 22 .…”

“…Such epistatic interactions may cause preferential selection or counterselection of certain genotypic combinations in a manner that would not be predicted from their individual effects. To study epistatic interactions between resistance mutations and resistance genes, we selected 11 different chromosomally barcoded mutants, adapted to resist different antibiotics, and 11 synthetic ARGs 10,22 . The mutants and ARGs were chosen to represent a wide range of mutational targets and biochemical resistance mechanisms (Supplementary Tables 1, 2).…”

“…Strains and plasmids. Twelve isolated colonies of chromosomally barcoded E. coli MG1655 K12 (DSMZ, DSM 18039) lineages were chosen from the endpoint of a large-scale adaptive evolution experiment based on the diversity of their mutation profile 22 . The mutations evolved as a response to the exposure of the strains to increasing drug concentrations.…”

Multiplexed competition in a synthetic squid light organ microbiome using barcode-tagged gene deletions

“…Barcode sequencing (BarSeq), in which each strain is labeled with a unique DNA sequence and sequenced using high-throughput next-generation sequencing, has been used successfully to track population dynamics in multiple systems, including in yeast genomic libraries, during Vibrio cholerae infection, and to track and phenotype laboratory-evolved Escherichia coli (50)(51)(52)(53)(54).…”

“…Directed evolution experiments have recently been applied to study colonization factors in V. fischeri(75). Barcode tagging also has applications in tracking specific lineages in long-term evolution experiments and analyzing microbial population dynamics during infection(50,53,54). The ease of tracking large numbers of individual V.fischeri strains using BarSeq enables long-term evolution experiments that track lineages through multiple rounds of colonization, growth, expulsion, and recolonization.…”