Storage polymer polyhydroxybutyrate promotes recovery and limits phenotypic heterogeneity in regrowth from carbon starvation in Methylobacterium extorquens

Bacteria in the wild often encounter long periods of starvation. Evolutionary adaptation to cycles of nutrient deficiency has led to metabolic and physiological strategies that prevent cell death and promote rapid recovery from starvation. A cellular factor shaping outcomes during carbon starvation is polyhydroxybutyrate (PHB), a widespread carbon polymer that helps maintain membrane integrity and promotes regrowth upon reintroduction to nutrients. The response to starvation is often highly heterogeneous across isogenic populations and confounded by environmental variables that mask underlying physiological mechanisms. Prior work has shown that hyper- or hypo-osmolarity greatly increases cell death, but little is known about how PHB and osmolarity jointly influence both population viability and the emergence of heterogeneity in periods of nutrient limitation. To investigate this, we subjected populations of Methylobacterium extorquens to carbon starvation across a range of osmolarities and analyzed the phenotypes of individuals regrowing from the combined stress. By resolving the distributions of key traits such as lag time and growth rate, we found that populations partition into subpopulations that appear either significantly or minimally impaired by the stresses presented. Osmolarity affected total survival, and surprisingly, populations maintained viability in ultra-pure water. PHB metabolism was beneficial for population viability across all conditions, and unexpectedly reduced the variance of populations in lag time and growth rate. These findings show that PHB aids survival and regrowth from starvation, and suggest that the restructuring of phenotypes in long-term stationary phase depends on PHB even though total osmotic sensitivity does not.