• Temperature adaptation of two Gram-negative bacterial strains isolated from Alpine lakes. • Non-linear response to temperature, relying mainly on non-hydroxy fatty acids. • The strains exhibit slight differences in temperature adaptation. 3-hydroxy fatty acids (3-OH FAs) produced by Gram-negative bacteria were recently proposed as promising temperature proxies. Nevertheless, the lipid adaptation mechanism of such microorganisms to temperature remains largely unexplored. Here, we investigated h