Thermal proteome profiling combined with a reverse genetics approach provides insights into the abundance and thermal stability of the global proteome of Escherichia coli. Recent developments in high-throughput reverse genetics(1,2) have revolutionized our ability to map gene function and interactions(3-6). The power of these approaches depends on their ability to identify functionally associated genes, which elicit similar phenotypic changes across several perturbations (chemical, environmental or genetic) when knocked out(7-9). However, owing to the large number of perturbations, these approaches have been limited to growth or morphological readouts(10). Here we use a high-content biochemical readout, thermal proteome profiling(11), to measure the proteome-wide protein abundance and thermal stability in response to 121 genetic perturbations in Escherichia coli. We show that thermal stability, and therefore the state and interactions of essential proteins, is commonly modulated, raising the possibility of studying a protein group that is particularly inaccessible to genetics. We find that functionally associated proteins have coordinated changes in abundance and thermal stability across perturbations, owing to their co-regulation and physical interactions (with proteins, metabolites or cofactors). Finally, we provide mechanistic insights into previously determined growth phenotypes(12) that go beyond the deleted gene. These data represent a rich resource for inferring protein functions and interactions.