Mutation analysis
Replifactory can run morbidostat experiments, functioning like a "fitness trainer" that applies gradually increasing stress levels on microorganisms. The stress level is controlled by adding a biocide such as antibiotics, alcohols or heavy metals. The genetic mutations that are responsible for adaptation provide insights about the underlying biological mechanisms - what modifications in the proteomic machinery of the cell make it resistant to a biochemical environment that was previously deadly. Such experiments are often used to study antimicrobial resistance - how pathogenic bacteria become immune to drugs that are supposed to treat infections. Understanding the molecular mechanisms of antimicrobial resistance can help develop evolutionarily informed treatment strategies.
Life finds a way, and we can see exactly how.
In this example we look at how E.coli adapts to Cobalt. The wild type strain showed significant growth disruption at 2mM, and after about 2 weeks of continuous evolution in a replifactory vial, the culture could grow in 60mM - a 30x increase! Both ancestral and adapted genomes were sequenced, and genomic analysis revealed 2 mutations, illustrated below:
The Adenine nucleotide at position 1075459 mutated to a Cytosine and the Guanine nucleotide at position 1545890 mutated to a Thymine. Everything else in the 4.7mb genome remained exactly the same, confirmed by full de novo assembly with >100x PacBio long read sequencing.
These mutations happen to be in genes encoding mgtA and corA on the reverse strand. In mgtA the TTC codon (phenylalanine) became TGC (cysteine) and in corA the TCG codon (Serine) became TAG - a stop codon that signals a halt to protein synthesis.
The corA protein forms a pentameric pore through which ions like Cobalt and Magnesium flow into the cell. The Cobalt influx activity enabled by corA is detrimental to cells stressed with far too much Cobalt. In the mutant variant, most of the 316 amino acid protein is not transcribed due to the stop codon that appeared at position 62, effectively resulting in a corA knockout. In replicate experiments two other corA knockouts were observed.
corA knockout: only a small part of the protein can be transcribed, effectively disabling any influx of Cobalt or Magnesium through the pore.
The mgtA protein transports Magnesium into the cell. The mutation identified above replaces phenylalanine 779 with cysteine, and in a replicate experiment threonine 815 is replaced by methionine. A 2024 paper described the structure of mgtA in detail, identifying the transmembrane magnesium binding site, composed of 4 amino acids: D780, N709, S776, and S813. The independent occurence of mutations in such proximity to the magnesium binding site suggests a functional relationship. The exact effect of these mutations wasn't yet determined, but one hypothesis is that they compensate for the decreased Mg influx following the corA knockout.
Another mutation in the replicate experiment is in the PhoP gene. PhoP is a transcription factor that upregulates mgtA under low magnesium concentration. The mutations in PhoP and mgtA each modify a single amino acid. The functional relationship between these proteins suggests that the magnesium metabolism machinery is important for cobalt adaptation.
mgtA mutations: the amino acids mutated in independent Cobalt adaptation experiments (red) are in the immediate vicinity of the transmembrane Mg-binding site (green)
Experimental evolution coupled with sequencing can identify proteins involved in adaptation and pinpoint functional areas responsible for their metabolic activity.
The code for the bioinformatics analysis and 3D mutation animations, as well as additional details are available on github.