Homologous recombination is an ubiquitous process that shapes genomes and repairs DNA damage. The reaction is classically divided into three phases: presynaptic, synaptic, and postsynaptic. In Escherichia coli, the presynaptic phase involves either RecBCD or RecFOR proteins, which act on DNA double-stranded ends and DNA single-stranded gaps, respectively; the central synaptic steps are catalyzed by the ubiquitous DNA-binding protein RecA; and the postsynaptic phase involves either RuvABC or RecG proteins, which catalyze branch-migration and, in the case of RuvABC, the cleavage of Holliday junctions. Here, we review the biochemical properties of these molecular machines and analyze how, in light of these properties, the phenotypes of null mutants allow us to define their biological function(s). The consequences of point mutations on the biochemical properties of recombination enzymes and on cell phenotypes help refine the molecular mechanisms of action and the biological roles of recombination proteins. Given the high level of conservation of key proteins like RecA and the conservation of the principles of action of all recombination proteins, the deep knowledge acquired during decades of studies of homologous recombination in bacteria is the foundation of our present understanding of the processes that govern genome stability and evolution in all living organisms.