Microbial phenotypic heterogeneity in response to a metabolic toxin: Continuous, dynamically shifting distribution of formaldehyde tolerance in Methylobacterium extorquens populations

Abstract

The funders had changing over time in response to growth conditions. We characterized this phenomenon using bulk liquid culture experiments, colony growth tracking, flow cytometry, single-cell timelapse microscopy, transcriptomics, and genome resequencing. Finally, we used mathematical modeling to better understand the processes by which cells change phenotype, and found evidence for both stochastic, bidirectional phenotypic diversification and responsive, directed phenotypic shifts, depending on the growth substrate and the presence of toxin.

EfgA is a conserved formaldehyde sensor that halts bacterial translation in response to elevated formaldehyde

“…Recent work demonstrating that formaldehyde stress tolerance is responsive to the environment suggests that cells can sense this toxic intermediate. Formaldehyde tolerance was found to be phenotypically heterogeneous across genetically identical individuals of M. extorquens PA1, whereby some cells could not even survive exposure to 1 mM formaldehyde when it was provided in the growth medium [33]. This is consistent with the paradoxical finding that many methylotrophs are unable to directly grow on formaldehyde even though they rapidly generate it during growth on more reduced C 1 compounds [25,33].…”

“…Given that mutations in efgA impact genetic resistance to formaldehyde, we questioned whether it was linked to phenotypic formaldehyde tolerance observed between genetically identical cells [33]. efgA was not represented amongst the differentially-expressed genes seen in the environmentally-responsive phenotypic variation in formaldehyde tolerance (Table S2).…”

“…Methylotrophs generate formaldehyde at high rate, yet several decades of work with methylotrophs had failed to reveal any proteins that sense and respond to its toxicity. By evolving M. extorquens to grow on 20 mM formaldehyde as a sole substrate, a concentration that was found to sterilize a wild-type culture in just two hours [33], we uncovered several genes that could permit growth. Notably, we report here that one of these, now named efgA for e nhanced f ormaldehyde g rowth, encodes a formaldehyde sensor that can act through inhibition of protein translation.…”

“…This method has a limit of detection of 1.65×10 −7 (an abundance of 34 CFU/mL is necessary to observe 1 cell per 30 μL plated, and the total cell population tested was 2×10 8 CFU/mL; therefore the least-abundant subpopulation that could be detected, disregarding the effects of Poisson distributions at lower λ, is one with an average frequency of 1.65×10 −7 within the total population). Although this assay measures the growth of bacterial colonies and not directly that of individual cells, it has been demonstrated to correlate well with single-cell methods of measuring formaldehyde tolerance distribution [33].…”

“…Formaldehyde tolerance was found to be phenotypically heterogeneous across genetically identical individuals of M. extorquens PA1, whereby some cells could not even survive exposure to 1 mM formaldehyde when it was provided in the growth medium [33]. This is consistent with the paradoxical finding that many methylotrophs are unable to directly grow on formaldehyde even though they rapidly generate it during growth on more reduced C 1 compounds [25,33]. Although some of the cells in populations were surprisingly sensitive to formaldehyde, other rare cells could grow at normal rates under conditions where the vast majority rapidly die.…”