Mg²⁺ deficiency affects heat shock resistance in Escherichia coli

While thermal processing remains central to ensuring food safety, sublethal heat intensities stemming from gradients or from milder processing efforts risk enabling microbial adaptation. In efforts to investigate the capacity of foodborne bacteria to acquire heat resistance, most studies have focused on stationary phase cells, although many regions of the food processing chain are permissive for growth. To determine how growth-competent cells can adapt to heat through evolution, we subjected exponentially growing Escherichia coli MG1655 cells to iterative heat cycling. Interestingly, while all evolved lineages readily acquired improved heat shock resistance, none of them seemed to do so via the anticipated upregulation of heat shock proteins. In contrast, whole genome sequencing indicated that most lineages acquired loss-of-function mutations in mgrB, encoding a negative regulator of the PhoPQ two-component signal transduction system. Further genetic analysis revealed that upregulation of the PhoPQ-dependent MgtA Mg²⁺ importer was causal for the heat shock resistance. Moreover, chemical Mg²⁺ supplementation phenocopied this resistance and even improved heat survival of several pathogenic enterobacteria. Our findings highlight cellular Mg²⁺ homeostasis as a key determinant of intrinsic heat resistance in several proliferating enterobacteria, and underscore how both adaptive mutations and Mg²⁺ availability can readily impose increased heat resistance in the food processing chain.